Oral dispersible vaccine comprising virosomes

ABSTRACT

The present disclosure is directed to oral vaccine dosage forms and processes for producing the oral vaccine dosage forms. The dosage forms include lipid-based vesicles (e.g., virosomes, liposomes) harboring an immunogenic amount of at least one vaccinal target molecule, with or without adjuvant. Specifically, Applicants discovered a combination of the composition of the liquid virosome concentrates, the composition of the base matrix for the solid dosage form formulation (excluding the virosome concentrate), and the manufacturing conditions for the dosage forms that can produce a freeze dried sublingual dosage form having physical robustness, particle and antigen integrity and stability.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/772,823, filed Nov. 29, 2018, the entire contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

This disclosure relates to an oral dosage form that induces mucosalimmunization.

More specifically, this disclosure relates to a freeze-dried orallydispersible vaccine containing virosomes. The project leading to thisapplication has received funding from the European Union's Horizon 2020research and innovation programme under grant agreement No 646122.

BACKGROUND OF THE INVENTION

Vaccines are traditionally delivered by intramuscular, intradermal, orsubcutaneous injections. These injections can produce strong systemicimmune responses, while the efficacy for triggering mucosal immuneresponses are variable and often weak or undetectable, particularly forsubunit vaccines. From the draining lymph nodes that have processed theinjected vaccine, antigen specific cytotoxic T cells (CTLs) andantibodies produced by B cells can migrate to different organs in thebody but their migration to the various mucosal tissues (e.g., genital,intestinal, respiratory) is often limited or not possible due toinadequate homing mucosal receptors and chemotaxis. However, theintranasal route that is also considered as a parenteral immunizationroute can trigger good mucosal immune responses in the respiratory,genital and intestinal tract that are sharing some interconnections,which is more accessible if the vaccine is delivered at the mucosalsite. Therefore, such parenteral vaccines may offer protection in somecases against mucosal pathogens.

Because most pathogens enter the body through mucosal tissues (oral,respiratory, genital, and intestinal tracts) and many of them onlyreplicate in the mucosal tissues, mucosal vaccination may optimallyinduce front line defense by inducing both innate (ex. NK cells) andadaptive (T and B cells) immune responses at the local and distantmucosal sites.

With resident mucosal defense, protection can be immediate with no delayin cell recruitment from the periphery, which allows to interfere moreefficiently with very early events of transmission and infection eventsprior pathogen spreading and in some cases, reservoir establishment.

Mucosal tissues and lymph nodes contain more than 90% of the immunecells. Moreover, mucosal antibodies represent about 80% of the totalbody antibody production. Thus, the local immune response by mucosalantibodies can act as a front line defense against mucosal infections(e.g., HIV-1, herpes viruses, rotaviruses, etc.) and entry acrossmucosal tissues for reaching other organs (e.g., HIV-1, hepatitis B,tuberculosis). In contrast, blood antibodies can act as a backup defenseonce the pathogens have crossed the mucosal defense or in synergy withmucosal antibodies and mucus environment. Blood antibodies primarily actas an efficient front line defense for dealing with pathogens enteringdirectly into the blood stream following mosquito bites (e.g., malaria,chikungunya, dengue, Zika, West Nile virus, yellow fever, etc.) oraccidental skin/mucosal injuries (e.g., Staphylococcus aureus,Pseudomonas aeruginosa).

Mucosal vaccine delivery (via the buccal, sublingual, nasal, oral, orvaginal mucosa) has received increasing interest as a means of inducinglocal and distant antibody immune response as well as systemic immuneresponse. In addition, mucosal vaccine delivery by solid dosage forms(e.g., buccal/sublingual tablets, oral tablets or capsules, vaginalinserts) can offer several advantages such as the potential for massimmunization, patient compliance, ease of use, product shelf lifestability, cold-chain independent capability. Furthermore, mucosalvaccine delivery can be suitable for patients that have needle injectionphobia and the patient can self-administrate the vaccine with adequateexplanations. The buccal/sublingual route has been used for many yearsto deliver drugs and small molecules to the bloodstream, but itsapplication as a means of mucosal delivery for vaccines has receivedlittle attention.

BRIEF SUMMARY OF THE INVENTION

Lipid-based vesicles can be used as drug, vaccine, or adjuvant deliverysystems, and combinations of thereof. Lipid-based vesicles can consistof one or several natural and/or synthetic lipids forming the basestructure (particle) and additional optional components (peptides,proteins, carbohydrates, nucleic acids, small molecules). Lipid-basedvesicles can range from the nanoparticle (approximately 20-200 nm) tothe sub-micrometer (approximately 200-800 nm) to the micrometer(approximately 800 nm-10 μm) scale.

Virosomes and liposomes belong to the lipid-based vesicle systems.Virosomes are unilamellar, and liposomes can be unilamellar, bilamellar,or multilamellar. Virosomes are a type of subunit vaccine that maycontain any enveloped virus derived protein that is used as startingmaterial for the formation of the lipid-based virosome particles. Thesevirosomes can be devoid of genetic material, non-replicative and notinfectious, which makes them suitable and safe for enteral andparenteral immunization, provided that they can be formulated in astabilized form. Virosomes may contain additional molecules such ashomologous (same pathogen origin) or heterologous target molecules(derived from a pathogen different from the starting virus material)under peptide, protein, and/or carbohydrate forms, nucleic acids,adjuvants, specific lipids and/or small molecules (drugs). Similar tovirosomes, liposomes can also be used as a vehicle for administration ofpharmaceutical drugs, vaccines, and adjuvants, but the lipid membranedoes not contain any native viral proteins. In some embodiments, theliposomes used herein can be proteoliposomes (i.e., liposomes withproteins).

Ideally, stabilization would either permit the vaccine storageindependent of the cold chain or allow the vaccine to support high andlow temperature excursions outside the recommended cold chain conditionswithout compromising the bioactivity of the product.

Applicants have discovered a freeze dried oral dispersible vaccinecontaining virosomes and the process of making such a vaccine that canpreserve the stability of the virosomes (both physically for theparticle structure and chemically for the target molecules). The productstability can be maintained during storage at ambient temperature (e.g.,about 25° C.), independent of the cold chain storage conditions, and mayalso support accidental freezing conditions (e.g., −about 4° C. to about−17° C.) as well as exposure to high temperatures present in warmcountries (e.g., 35° C. to 45° C.). The freeze dried sublingual dosageforms disclosed herein that contain the virosomal vaccine can inducemucosal immunity, and may also elicit systemic immune responses.Specifically, the combination of the composition of the liquid virosomeconcentrate and its buffer, the composition of the liquid base matrixfor solid dosage form (excluding the virosome concentrate), and themanufacturing conditions for generating solid dosage forms under freezedried sublingual tablets can generate a solid vaccine form that hasproper physical attributes for sublingual tablets, with preservedvirosomes and target molecule integrity, harboring stability duringstorage under different environmental conditions. Administering stablevirosome solid dosage forms can be suitable for sublingual mucosalimmunization.

Virosomes belong to the enveloped virus like particle (“VLP”) family, asthey have lipid membranes containing viral membrane proteins derivedfrom the starting purified viruses. As VLPs, virosomes can closely mimicthe virus particle architecture, composition, antigen membrane surfacepresentation, with or without functional activities of the native viralenvelope. Virosomes may comprise reconstituted viral membranes,generally obtained by purification of solubilized membrane proteins andlipids from enveloped viruses with solubilization agent, followed byaddition of natural or synthetic lipids and antigens, with or withoutadjuvants, and removal of said solubilization agent from the mixture,leading to lipid bilayer formation with the proteins protruding fromthem. Antigens can be native proteins as well as recombinant orsynthetic proteins or peptides. Virosomes may also be formed first, andthen modified by the covalent or non-covalent modification of theirmembranes to contain adjuvants and other molecules. A characteristicfeature of virosomes is that they can closely mimic the composition,surface architecture and functional activities of the native viralenvelope. In some cases, when the induction of CD8+ T cells is part ofthe vaccine strategy, a particularly important characteristic of saidvirosomes is the preservation of the hemagglutinin (HA) receptor-bindingwith its fusion membrane activity. If the vaccine strategy focuses onthe induction of antigen specific antibodies, then the HA fusionactivity is not absolutely required but the presence of HA as excipientis still important, as it can provide T cell help, particularly forsmall antigens and peptides that maybe devoid of such properties.Although specific vaccines are mentioned in this disclosure, allvaccines that use virosomes as a delivery platform are contemplated bythis disclosure. Influenza virosome containing HIV antigens (P1 andrgp41) and TLR 7/8 adjuvant can be used as model virosome concentratesfor the vaccines disclosed herein. The influenza virosomes with HIVantigen rgp41 have been shown to be capable of eliciting immuneresponses in various animal studies and in a Phase 1 clinical study viathe intranasal and intramuscular route. In such studies, the virosomeformulations were under liquid form administered by needle or nasalliquid spray, which were susceptible to show limited shelf lifestability at 4° C. due to chemical modifications of the ActivePharmaceutical Ingredients (“APIs”). To prevent or reduce such APIchemical modifications often present in aqueous environments, developingsolid vaccine forms with low moisture content was identified as apromising approach.

Applicants were able to overcome various problems in producing a solidvaccine form as the freeze dried oral dispersible vaccines disclosedherein. Specifically, the influenza-based virosome concentrates thatcontain the virosomes carrying antigens and adjuvants may be supplied asa suspension in a buffer salt solution. To produce the freeze driedvaccine dosage forms, robust and porous structures within the tabletsshould be produced during manufacture and sustained during subsequentstorage. The presence of buffer salts in the virosome concentrates canpresent a challenge in the manufacture of such a robust and porousstructure. As such, Applicants discovered an appropriate amount ofexcipients in conjunction with manufacturing conditions to enable theformation of a robust dosage form that does not collapse or partiallycollapse during the freeze drying process.

In addition, the freezing, annealing, and freeze drying process may alsodamage the integrity of the virosome particles. This damage to thevirosomes should be minimized such that sufficient virosome particlesremain in the freeze dried product such that a sufficient amount of themcan cross the sublingual mucosal membrane for being processed by theimmune cells to induce mucosal immune responses, that maybe alsosupported by a systemic immune responses. As such, Applicants were ableto balance the excipient usage and freeze drying process to maintainboth the virosome immunogenicity (e.g., intact virosomes with limitedpresence of clusters) and good dosage form properties (e.g., orallydisintegrating sublingual tablets).

In some embodiments, an oral solid vaccine dosage form includeslipid-based vesicles comprising an immunogenic amount of at least onetarget molecule; 5-20 wt. % of at least one cryo-lyoprotectant; 25-40wt. % of a matrix former; and 40-55 wt. % of a structure former. In someembodiments, the lipid-based vesicles are virosomes or proteoliposomes.In some embodiments, the dosage form comprises 10-15 wt. % of at leastone cryo-lyoprotectant. In some embodiments, the at least onecryo-lyoprotectant comprises trehalose. In some embodiments, the dosageform comprises 33-37 wt. % of the matrix former. In some embodiments,the matrix former comprises gelatin. In some embodiments, the gelatincomprises fish gelatin. In some embodiments, the fish gelatin is highmolecular weight fish gelatin. In some embodiments, the dosage formcomprises 45-50 wt. % of the structure former. In some embodiments, thestructure former comprises mannitol. In some embodiments, the virosomesare derived from the influenza virus membrane or other envelopedviruses. In some embodiments, the at least one target molecule ispresent on the virosome. In some embodiments, the at least one targetmolecule comprises an HIV-1 envelope derived antigen. In someembodiments, the HIV-1 envelope derived antigen comprises HIV-1 PIpeptide and/or HIV-1 recombinant gp41. In some embodiments, thevirosomes comprise adjuvant. In some embodiments, the dosage formfacilitates oral cavity uptake of the at least one target molecule. Insome embodiments, the dosage form disintegrates within 180 seconds afterbeing placed in the oral cavity. In some embodiments, the dosage formdisintegrates within 90 seconds after being placed in the oral cavity.In some embodiments, the dosage form disintegrates within 60 secondsafter being placed in the oral cavity. In some embodiments, the dosageform disintegrates within 30 seconds after being placed in the oralcavity. In some embodiments, an immune response is induced whenadministered to a patient by placement in the oral cavity. In someembodiments, placement in the oral cavity is placement on or under thetongue or in the buccal or pharyngeal region.

In some embodiments, a method of inducing an immune response in apatient includes placing any of the dosage forms above in an oral cavityof a person in need of the immune response. In some embodiments,placement in the oral cavity is placement on or under the tongue or inthe buccal or pharyngeal region.

In some embodiments, a method of forming an oral solid vaccine dosageform includes dosing a liquid virosome formulation into a preformedmold, wherein the virosome formulation comprises: lipid-based vesiclescomprising an immunogenic amount of at least one target molecule, 1-5wt. % a cryo-lyoprotectant, 4-8 wt. % of a matrix former, and 5-10 wt. %of a structure former; freezing the dosed viro some formulation at atemperature of −60° C. to −90° C.; annealing the frozen virosomeformulation by holding it at a temperature of less than −15° C. for 3-9hours; and freeze-drying the annealed virosome formulation to form thedosage form. In some embodiments, the dosed virosome formulation isfrozen at a temperature of −60° C. to −90° C. for a duration of about1-5 minutes. In some embodiments, freeze-drying the annealed virosomeformulation comprises a first step of holding the annealed virosomeformulation at a temperature of −10° C. to −20° C. for 20-28 hours and asecond step of holding the annealed virosome formulation at atemperature of −5° C. to about −15° C. for 14-22 hours. In someembodiments, the freeze-drying occurs at a pressure of less than 600mbar. In some embodiments, the virosome formulation has a pH of about6.5-8. In some embodiments, the cryo-lyoprotectant comprises trehalose.In some embodiments, the matrix former comprises gelatin. In someembodiments, the gelatin comprises fish gelatin. In some embodiments,the fish gelatin is high molecular weight fish gelatin. In someembodiments, the structure former comprises mannitol. In someembodiments, the lipid-based vesicles are derived from the influenzavirus or respiratory syncytial virus. In some embodiments, the at leastone target molecule comprises an HIV-1 envelope derived antigen. In someembodiments, the HIV-1 envelope derived antigen comprises HIV-1 PIpeptide or HIV-1 recombinant gp41. In some embodiments, the lipid-basedvesicles comprise adjuvant.

In some embodiments, a method of forming an oral solid vaccine dosageform includes: dosing a liquid virosome formulation into a preformedmold, wherein the virosome formulation comprises: (1) 20-50 wt. % of avirosome concentrate, wherein the virosome concentrate comprises:virosomes comprising an immunogenic amount of at least one targetmolecule; 2-10 wt. % of a cryo-lyoprotectant; and 60-200 mM of a buffersystem; (2) 4-8 wt. % of a matrix former; and (3) 5-10 wt. % of astructure former; freezing the dosed virosome formulation at atemperature of −60° C. to −90° C.; annealing the frozen virosomeformulation by holding it at a temperature of less than −15° C. for 3-9hours; freeze-drying the annealed virosome formulation to form thedosage form. In some embodiments, the buffer system comprisesHEPES-Sodium Chloride.

Additional advantages will be readily apparent to those skilled in theart from the following detailed description. The examples anddescriptions herein are to be regarded as illustrative in nature and notrestrictive.

All publications, including patent documents, scientific articles anddatabases, referred to in this application are incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication were individually incorporated by reference. If adefinition set forth herein is contrary to or otherwise inconsistentwith a definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are described with reference to the accompanyingfigures, in which:

FIG. 1 illustrates a flow chart overview for producing a vaccine dosageform disclosed herein.

FIG. 2 illustrates a flowchart from matrix formulation to finalsublingual tablets for producing a vaccine dosage form disclosed herein.

FIG. 3A is a schematic representation with a picture of a fully wettedtablet.

FIG. 3B is a schematic representation with a picture of a tablet withhard lumps.

FIG. 3C is a schematic representation with a picture of a tablet with afilm of collapsed formulation matrix that forms at the surface of thefreeze dried tablet (skin).

FIG. 4 is a photo of immunoblots showing anti-P1 and anti-rgp41 specificantibody binding to virosomes-P1 and virosomes-rgp41 from reconstitutedZydis® sublingual tablets stored at 5° C., 25° C., and 40° C. over 1 and3 months (analysis of Example 2 disclosed herein).

FIG. 5 illustrates the immunogenicity of P1 and rgp41 antigens of theliquid virosome concentrates and freeze-dried sublingual tabletscontaining virosomes, both stored at 4° C. and 40° C. over time.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are pharmaceutical compositions for lipid-basedvesicles (e.g., virosomes or VLPs) and methods of preparing thesepharmaceutical compositions. Specifically, the present disclosurerelates to freeze dried orally dispersible or disintegrating dosageforms that can preserve the stability of the VLPs (i.e., structuralintegrity and antigen chemical stability), can be stored independent ofcold chain storage conditions and can also support accidental freezingconditions as well as exposure to high temperatures present in warmcountries. The dosage forms can also retain the VLP's physical andchemical attributes making it suitable for sublingual delivery to inducemucosa immunization.

Applicants were able to formulate and optimize the amount of matrixformer and structure former to have a formulation that can address theuse of a high loading of a virosome concentrates containing buffersystems and cryo-lyoprotectants. In conjunction with the excipientadjustments, the manufacturing parameters of the dosage forms wereoptimized. Specifically, the annealing time was optimized to maximizethe mannitol crystallization that imparts dosage form robustness andminimize the virosome damage. In addition, the freeze-drying conditionswere optimized to minimize damage to the virosome particles as well asminimize structural collapse during freeze-drying.

Lipid Based Vesicle Systems

Lipid-based vesicles can be used as drug, vaccine, or adjuvant deliverysystems, and combinations of thereof. Lipid-based vesicle systems canconsist of one or several natural and/or synthetic lipids (ex.phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,phosphatidylinositol, phosphatidylglycerol, cholesterol, sphingolipidsand their derivatives) forming the vesicle membrane (particle) andadditional optional components (peptides, proteins, carbohydrates, smallmolecules). Lipid-base vesicles range from the nanoparticle (about20-200 nm) to the sub-micrometer (about 200-800 nm) to the micrometer(about 800 nm-10 μm) scale.

A lipid-based vesicle/particle may comprise a unilamellar, bilamellar,or a multilamellar lipid bilayer vesicle. In some embodiments, alipid-based vesicle/particle can include a lipid bilayer comprisinglipids chosen from natural and/or synthetic lipids. Such lipids may beused to better mimic the pathogen membrane and lipid raft microdomainsin order to improve antigen membrane anchorage, antigen presentation,and/or folding for optimal epitope exposure. The lipid-basedvesicle/particle may harbour membrane anchored antigen and/or adjuvantexposed at the surface of the particle or pointing toward inside theparticle or having a random orientation. Antigen and/or adjuvant canalso be encapsulated inside the lumen of the lipid-basedvesicle/particle.

Virosomes are lipid-based vesicles in vitro assembled, in a cell-freesystem manner, forming enveloped VLPs that belong to the subunit vaccinecategory. Virosome lipid membranes as carrier can be derived from anyenveloped virus and consequently, contain at least native viral membraneproteins from the starting virus. These virosomes are devoid of geneticviral material, can't replicative and are not infectious, which makesthem suitable and safe for systemic and mucosal immunization. Inaddition, virosomes may contain additional molecules such as antigens(e.g. peptides, proteins, carbohydrates, nucleic acids), adjuvants,specific lipids and/or small molecules (drugs), which can be anchored atthe virosomes surface and/or entrapped inside the virosome lumen.

Liposomes are also lipid-based vesicles forming a type of subunitvaccine that can be formed as vesicles having at least one lipidbilayer, which don't contain proteins derived from natural viralmembrane of enveloped viruses. Such lipid-based particles also in vitroassembled are devoid of genetic material and can be suited for systemicand mucosal application. In addition, liposomes may contain additionalmolecules such as antigens (peptides, proteins and/or carbohydrates,nucleic acids), adjuvants, specific lipids and/or small molecules(drugs), which can be anchored at the liposome surface and/or entrappedinside the liposome lumen. In some embodiments, the liposomes usedherein can be proteoliposomes (i.e., liposomes with proteins).

The lipids used in the dosage forms described herein can belong to thecationic lipids, glycolipids, phospholipids, glycerophospholipids,galactosylceramid, sphingolipids, cholesterol and derivatives thereof.Phospholipids may include, but are not limited to, phosphatidylcholine,sphingomyelin, phosphatidylethanolamine, phosphatidylserine,phosphatidylglycerol, phosphatidic acid, cardiolipin, andphosphatidylinositol with varying fatty acyl compositions.

In addition, lipids may be chosen from DOTMA(N-[1-(2,3-dioleylaxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, DODAC(N,N-dioleyl-N,N,-dimethylammonium chloride), DDAB(didodecyldimethylammonium bromide) and stearylamine or other aliphaticamines and the like.

Virosome Formulation

Virosome Concentrate

FIG. 1 illustrates a flow chart for a method 100 of producing a vaccinedosage form disclosed herein. At step 101, a liquid virosome concentratecan be mixed with a premixture of base matrix formulation to form aliquid virosome formulation suitable for freeze-drying. Applicants havefound that when a composition of a virosome concentrate is combined witha specific composition of a base matrix formulation and prepared inconjunction with a set of manufacturing conditions optimized to preservesufficient virosomes with the required particle characteristics, thevaccine bioactivity can be maintained. The bioactivity of the virosomeparticles can be dependent on the conformational integrity of theparticles and quality of the antigenic molecules that are associatedwith its unilamellar phospholipid membrane.

In some embodiments, the virosome concentrate can be a liquid virosomeconcentrate. The virosome concentrate includes at least one virosomepopulation. A virosome population can be virosomes containing a givendrug, or virosomes acting as vaccine delivery vehicle harboring vaccinalantigens and virus derived proteins (e.g., HA if the virosomes arederived from influenza virus).

In some embodiments, the virosomes can be derived from an influenzavirus for generating influenza virosomes as enveloped VLPs acting ascarrier for heterologous vaccinal antigens (ex. HIV antigens anchored oninfluenza derived virosomes) or from another enveloped virus like therespiratory syncytial virus (“RSV”), In some embodiments, the envelopedviruses like the RSV, the Sendai virus, Semliki Forest virus (SFV),vesicular stomatitis virus (VSV), or Sindbis can be used for generatingthe corresponding RSV-virosomes, Sendai-virosomes, SFV-virosomes,VSV-virosomes or Sindbis-virosome for homologous vaccinal antigendisplayed (ex. native RSV antigens on virosomes derived from RSV). Insome embodiments, the virus based virosomes can be derived from anyenveloped virus. In some embodiments, the virus based virosomes can bederived from DNA viruses including, but not limited to, Herpesviruses,Poxyviruses, and Hepadnaviruses. In some embodiments, the virus basedvirosomes can be derived from RNA viruses including, but not limited to,Flavivirus, Togavirus, Coronavirus, Hepatitis D, Orthomyxovirus,Paramyxovirus, Rhabdovirus, Bunyavirus, and Filovirus. In someembodiments, the virus based virosomes can be derived from retroviruses.

In some embodiments, an influenza virus based virosome can be formedaccording to the different processes described in Influenza virosomes asvaccine adjuvant and carrier system; Moser C. et al, Expert review,779-791 (2013); WO2004110486; WO2004071492; WO2007099446; WO2016039619;EP2058002; and WO2016039620, which are hereby incorporated herein byreference in their entirety. In addition, the various patents and otherpublications listed in the previously cited references are alsoincorporated herein by reference in their entirety. As such, thisapplication is not limited to a specific process for virosomepreparation. As such, the reference described above is simply an exampleof virosome preparation. This application applies to all lipid-basedparticles such as but not limiting to virosomes, VLPs, and nanoparticlepreparation, including liposomes with antigens.

Although this section describes a liquid virosome concentrate, thevirosome can be replaced with a liposome to form a liquid liposomeconcentrate. As such, the components in the virosome concentrate(besides the virosomes themselves) can equally be applied to a liposomeconcentrate.

The virosome (e.g., influenza derived virosome) can have viral proteinson its surface. In some embodiments, the protein can be hemagglutinin(“HA”) and/or neuraminidase (“NA”). In some embodiments, the liquidvirosome concentrate includes viral membrane proteins (e.g. HA) presentin a concentration detectable by state of the art analytical assays. Insome embodiments, the viral membrane proteins are about 10-300 μg/mL orabout 5-150 μg/mL of the liquid virosome concentrate, when usinginfluenza virosomes as carrier of heterologous vaccinal antigens. Forinfluenza virosomes designed for inducing CTL responses, the HAconcentration can range at about 150-800 μg/mL or about 75-400 μg/mL. Insome embodiments the concentration of viral membrane proteins can begreater than the example concentrations listed above.

The virosome concentrate can also include lipids. The lipids used in thevirosome concentrate can belong to the cationic lipids, glycolipids,phospholipids, glycerophospholipids, galactosylceramid, sphingolipids,cholesterol and derivatives thereof. Phospholipids may include, inparticular, phosphatidylcholine, sphingomyelin,phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol,phosphatidic acid, cardiolipin, and phosphatidylinositol with varyingfatty acyl compositions. In addition, lipids may be chosen from DOTMA(N-[1-(2,3-dioleylaxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, DODAC(N,N-dioleyl-N,N,-dimethylammonium chloride), DDAB(didodecyldimethylammonium bromide) and stearylamine or other aliphaticamines and the like. In some embodiments, the liquid virosomeconcentrate can include about 0.1-10 mg/mL, about 0.3-8 mg/mL, or about0.5-5 mg/mL of lipids.

The virosomes can contain target molecules (e.g., vaccinal antigens)with or without adjuvants. Antigens can be soluble and entrapped insidethe virosome lumen or covalently or non-covalently anchored onvirosomes, which can be peptides, proteins, polysaccharides, whole orpartial fragments or extracts of bacterial cells, viral particles, ornucleic acids, or can be derived from a parasite, such as a protozoan orworm, which causes disease, or combinations thereof, or derived from anyplant, animal or human cells or cell lines. Any antigen known in the artcan be suitable for use with the virosomes, including those commerciallyavailable, or made by purification of preparations of a pathogen orcancer cell or non-transformed cell (native proteins), recombinantlyexpressed, or produced synthetically by standard manufacture. Methodsfor generating suitable antigens for incorporation into the virosomesare known in the art, and any of the known methods may be used asdisclosed herein.

The target molecule(s) is included in the virosomes of the liquidvirosome concentrate and in the subsequent dosage forms disclosed hereinin an amount, which is sufficient to render it immunogenic when providedin a dosage form. The “immunogenic amount” is defined as the amountappropriate to provoke a desired immune response. A person of skill inthe art can readily determine the immunogenic amount for a given diseaseor infection based on, among other facts, route of immunization, age andweight of the patient to whom the dosage form will be administered.

In some embodiments, the liquid virosome concentrate may include about1-2000 μg/mL of target molecule. The target molecule may be a peptide, aprotein, a carbohydrate, a nucleic acid, or a small molecule, or acombination thereof. The target molecule may function as an antigen(e.g., vaccinal antigen), a drug, a diagnostic molecule, an analyticalsensor, or a combination thereof. In some embodiments, the liquidvirosome concentrate can include about 1-1000 μg/mL of one targetmolecule and about 1-1000 μg/mL, of a second target molecule. Afterdownstream processing into solid dosage forms, each tablet may containabout 0.01 to 250 μg of each target molecule.

In more preferred embodiments, the liquid virosome concentrate mayinclude about 25-500, about 50-500 μg/mL, about 25-225 μg/mL, about25-200 μg/mL, about 50-450 μg/mL, about 50-400, about 100-400 μg/mL,about 100-400 μg/mL, about 100-200 μg/mL, or about 200-400 μg/mL of atleast one target molecule (e.g., antigen). In some embodiments, theliquid virosome concentrate can include about 1-500 μg/mL, about 25-500μg/mL, about 50-450 μg/mL, about 50-400 μg/mL, about 25-250 μg/mL, about25-200 μg/mL, about 50-250 μg/mL, about 75-225 μg/mL, or about 100-200μg/mL of one target molecule and about 50-450 μg/mL, about 50-400 μg/mL,about 25-225 μg/mL, about 25-200 μg/mL, about 100-500 μg/mL, about150-450 μg/mL, about 175-425 μg/mL, or about 200-400 μg/mL of a secondtarget molecule.

The dosage forms disclosed herein can be used to deliver therapeutic orprophylactic vaccines to prevent or reduce symptoms related to allergiesor infection, tumor development and spreading, pathogen transmission,cell infection, pathogen load, after induction of B cells (antibodies)and/or relevant T cell subsets (ex, Th1, Th2, Th16, Thf, Tc1, Tc2, Tc3,Treg, others), which depends on the virosome formulations. To that end,the target molecules can provide protection against the followingrepresentative list of diseases which is not exhaustive: influenza,tuberculosis, meningitis, hepatitis, whooping cough, polio, tetanus,diphtheria, malaria, cholera, herpes, typhoid, HIV/AIDS, measles, lymedisease, travellers' diarrhea, hepatitis A, B and C, otitis media,dengue fever, rabies, parainfluenza, rubella, yellow fever, dysentery,legionnaires disease, toxoplasmosis, q-fever, hemorrhagic fever,Argentina hemorrhagic fever, caries, chagas disease, urinary tractinfection caused by E. coli, pneumoccoccal disease, mumps, chikungunya,cancer, allergies, and combinations thereof. In addition, the targetmolecules may provide protection against disease caused by thefollowing, non-exhaustive list of causative organisms: Vibrio species,Salmonella species, Bordetella species, Haemophilus species,Toxoplasmosis gondii, Cytomegalovirus, Chlamydia species, Streptococcalspecies, Norwalk Virus, Escherischia coli, Helicobacter pylori,Rotavirus, Neisseria gonorrhae, Neisseria meningiditis, Adenovirus,Epstein Barr virus, Japanese Encephalitis Virus, Pneumocystis carini,Herpes simplex, Clostridia species, Respiratory Syncytial Virus,Klebsiella species, Shigella species, Pseudomonas aeruginosa,Parvovirus, Camylobacter species, Rickettsia species, Varicella zoster,Yersinia species, Ross River Virus, J. C. Virus, Rhodococcus equi,Moraxella catarrhalis, Borrelia burgdorferi, Pasteurella haemolytica,and combinations thereof. In addition or alternatively, the targetmolecule may provide protection or treatment against allergies (i.e.,virosomes containing allergens), cancer (e.g., tumor antigens,antibodies, anti-cancer drugs, nucleic acid), and other types ofconditions.

Veterinary applications of the present disclosure are also contemplated.Accordingly, the target molecules can provide protections against thefollowing non-exhaustive list of veterinary diseases: coccidiosis,Newcastle disease, enzootic pneumonia, feline leukemia, atrophicrhinitis, erysipelas, foot and mouth disease, swine, pneumonia, andother disease conditions and other infections affecting companion andfarm animals, and combinations thereof.

In some embodiments, the virosome contains at least one vaccinal antigenin addition to the viral proteins present in the reconstituted membrane.In some embodiments, the vaccinal antigen can be HIV-1 P1 peptide and/orHIV-1 recombinant gp41.

As stated above, the virosomes of the liquid virosome concentrate mayalso contain, or be admixed with, an adjuvant. Virosomes and othersubunit vaccines may require an adjuvant for improving the immuneresponse, resulting in accelerated and enhanced production of antibodiesand T cells, while sustaining also the immunological memory. To beeffective, it is preferable to have an immune response associated withthe generation of a memory response that provides long lastingprotection from the specific disease. The adjuvant also can allow tolower the antigen dose (dose sparing) and increase the breadth of thedesired immune response. Once exposed to the antigens, the immune systemcan “remember” it and during re-exposure, the immune response is muchfaster. The effectiveness of an adjuvant to enhance an immune responsecan be independent from the antigen with which it is being combined, asadjuvant alone can trigger unspecific immune responses and may lead toautoimmune side effects if strong cell activation is achieved in theabsence of antigen. However, when the antigen and adjuvant arephysically link together, they all can co-migrate to the same site uponinjection, which favors antigen specific immune activation with lowerunspecific inflammatory responses. Suitable adjuvants include, but arenot limited to: Toll-like receptor (TLR) agonists, inflammasomeagonists, nucleotide-binding and oligomerization domain (NOD)—likereceptors (NLRs) agonists, more specifically non-toxic bacterialfragments, cholera toxin (and detoxified forms and fractions thereof),chitosan, heat-labile toxin of E. coli (and detoxified forms andfractions thereof), lactide/glycolide homo.+−.and copolymers (PLA/GA),polyanhydride, e.g., trimellitylimido-L-tyrosine, DEAE-dextran, saponinscomplexed to membrane protein antigens (immune stimulatingcomplexes—ISCOMS), bacterial products such as lipopolysaccharide (LPS)and muramyl dipeptide, (MDP), liposomes, cochleates, proteinoids,cytokines (interleukins, interferons), genetically engineered livemicrobial vectors, non-infectious pertussis mutant toxin,neurimidase/galactose oxidase, and attenuated bacterial and viral toxinsderived from mutant strains, and combinations thereof. A suitable amountof an adjuvant can be readily determined by one of ordinary skill in theart.

In some embodiments, the virosomes can harbor the adjuvant 3M-052 (aTLR7/8 agonist supplied by 3M). In some embodiments, the liquid virosomeconcentrate may contain 3M-052 adjuvant in the range of about 8-140μg/mL, about 4-70 μg/mL, about 1-60 μg/mL, and 0.01 to 16 μg per tablet.

In some embodiments, the liquid virosome concentrate can include atleast two different virosome populations, each harboring at least oneantigen with or without adjuvant. In some embodiments, these twodifferent virosome populations can have different antigens, but the sameadjuvant (e.g. virosome-P1/3M-052 mixed with virosomes-rgp41/3M-052). Insome embodiments, these two different virosome populations can havedifferent antigens, but with different adjuvant (e.g.virosome-P1/Adjuvant A mixed with virosomes-rgp41/Adjuvant B).

In some embodiments, the virosome concentrate can include at least twodifferent antigens per virosome, with or without adjuvant (e.g. virosomeharboring both P1 and rgp41 antigens, with or without adjuvant).

The liquid virosome concentrate can also include a buffer system. Insome embodiments, the virosomes can be suspended in the buffer system.The buffer system can maintain the physical integrity and chemicalstability of the virosomes in the virosome concentrate. In someembodiments, the virosomes are suspended in a buffer system to maintaina target pH of about 6-9, about 6.5-8, about 7-8, about 7.2-7.6, about7.3-7.5, or about 7.4. In addition, the buffer system can stabilize thevirosomes when it is in a liquid form at storage temperature of about2-8° C.

Suitable buffer system include, without limitation, HEPES-SodiumChloride (HN) buffers, HEPES-Sodium Chloride-EDTA (HNE) buffers,phosphate buffer systems (PBS), or combinations thereof. In someembodiments, the buffer system can be about 5-1000 mM, about 60-200 mM,about 100-300 mM, about 125-275 mM, about 150-250 mM, about 175-225 mMabout 180-210 mM, about 185-200 mM, about 185-195 mM, or about 190-195mM in the virosome concentrate. If the buffer system is HEPES-SodiumChloride in the virosome concentrate, the sodium chloride can be about5-1000 mM, about 50-150 mM, about 125-175 mM, about 130-160 mM, about130-150 mM, about 135-145 mM, or about 140-145 mM in the virosomeconcentrate. If the buffer system is HEPES-Sodium Chloride in thevirosome concentrate, the HEPES can be about 1-200 mM, about 10-75 mM,about 10-50 mM, about 25-75 mM, about 30-70 mM, about 40-60 mM, about45-55 mM, or about 48-52 mM in the virosome concentrate.

The liquid virosome concentrate can also include at least onecryo-lyoprotectant. Virosomes can be damaged during the freezing and/orfreeze drying steps of producing the dosage forms disclosed herein. Assuch, a cryo-lyoprotectant can be included into the virosome concentrateto improve virosome preservation during the freezing and/or freezedrying steps. Examples of cryo-lyoprotectants include, but are notlimited to, polyols such as trehalose, sugars such as sucrose, and aminoacids such as lysine, oligosaccharides such as inulin (a medium chainoligosaccharide), or combinations thereof. The cryo-lyoprotectants usedcan be inert to be suitable for vaccine formulation. The liquid virosomeconcentrate can include about 1-20% w/w, about 1.5-10% w/w, about 2-10%w/w, about 4-10% w/w, about 2-9% w/w, about 2-5% w/w, about 3-8% w/w,about 3.5-8% w/w, about 3.5-7% w/w, about 4-8% w/w, or about 5-7% w/wthe cryo-lyoprotectant.

The virosome concentrate can be about 1-75% w/w, about 10-65% w/w, about15-60% w/w, about 20-55% w/w, about 20-50% w/w, or about 25-50% w/w ofthe virosome formulation. In some embodiments, the virosome concentratecan be about 15-35% w/w, about 20-30% w/w, about 23-27% w/w, or about25% w/w of the virosome formulation.

Base Matrix Formulation

The base matrix formulation is what helps provide the structure of thefinal dosage form. As such, the base matrix formulation can include amatrix former. The matrix former can provide the network structure ofthe dosage form that imparts strength and resilience during handling.Suitable matrix formers can include, without limitation, gelatin,starch, or combinations thereof. Additional matrix formers can be foundin EP 2624815 B 1, which is herein incorporated by reference in itsentirety. The gelatin can be fish gelatin, bovine gelatin, porcinegelatin, or combination thereof. Each of the gelatins can have differentgelling characteristics. The extent a gelatin solution forms a gel candependent on the concentration of the gelatin and the temperature of thegelatin solution. A solution of bovine gelatin tends to gel attemperatures of less than 18° C. and thus can be considered a gellinggelatin. In contrast, fish gelatin can remain in solution attemperatures as low as 10° C. and thus can be considered a non-gellinggelatin. In some embodiments, the gelatin can be a low endotoxin gelatinsuch as one sourced or one produced according to the process disclosedin Provisional Application No. 62/640,394, which is hereby incorporatedby reference in its entirety. In some embodiments, the amount of matrixformer in the virosome formulation can be about 1-15% w/w, about 2-12,about 3-10% w/w, about 4-8% w/w, about 4-6%, about 5-7% w/w, or about 6%w/w.

The temperature at which the virosome formulation is dosed can be as lowas 10-18° C. As such, a formulation using bovine gelatin alone may notbe dosed at these low temperatures. However, a combination of bovinegelatin and another type of gelatin (e.g., fish gelatin) can be used.Applicants discovered that fish gelatin can provide a freeze-driedtablet with robust matrix structure and a disintegration time of about30-180 or 30-60 seconds that is desirable to impact sufficient contacttime with the oral mucosa. In addition, the fish gelatin can providefreeze dried dosage forms of good physical attributes for formulationcompositions that contain a high loading of soluble component likebuffer salts, such as the amounts disclosed herein.

In some embodiments, the fish gelatin can be high molecular weight fishgelatin, standard molecular weight fish gelatin, or combinationsthereof. High molecular weight fish gelatin is defined as a fish gelatinin which more than 50% of the molecular weight distribution is greaterthan 30,000 Daltons. Standard molecular weight fish gelatin is definedas fish gelatin in which more than 50% of the molecular weightdistribution is below 30,000 Daltons.

In some embodiments, the matrix former can also serve as a stabilizerfor the antigens as well as a muco-adhesive. In addition, starch canalso serve as an immune-stimulant excipient.

The base matrix formulation can also include a structure former.Suitable structure formers can include sugars including, but not limitedto, mannitol, dextrose, lactose, galactose, cyclodextrin, orcombinations thereof. The structure former can be used in freeze dryingas a bulking agent as it crystallizes to provide structural robustnessto the freeze-dried product. Soluble excipients such as buffer salts andtrehalose in the virosome formulation can inhibit its crystallization.An extended annealing time is typically used to allow forcrystallization. However, the presence of these soluble excipients canalso cause melting of the frozen product during annealing. As such,Applicants discovered a balance between the amount of structure former,buffer salts, and cryo-lyoprotectant and the annealing conditions (i.e.,temperature and time). In some embodiments, the amount of structureformer in the virosome formulation can be about 1-20% w/w, about 3-15%w/w, about 4.5-10% w/w, about 4.5-8% w/w, about 5-10% w/w, about 6-10%w/w, about 7-9% w/w, or about 8% w/w. Applicants discovered that atvalues below 4.5% w/w of the structure former, some microstructuralcollapse may occur during freeze drying resulting in poordispersion/disintegration of the freeze-dried dosage form. As such, ahigher amount of the structure former was found to minimize or eliminatethe microstructural collapse without drastically affecting virosome.

In addition, the base matrix formulation can also include acryo-lyoprotectant. Examples of cryo-lyoprotectants include, but are notlimited to, polyols such as trehalose, sugars such as sucrose, and aminoacids such as lysine, oligosaccharides such as inulin (a medium chainoligosaccharide), or combinations thereof. A cryo-lyoprotectant can beused to protect the virosome from damage during subsequent freezing andfreeze drying. However, the addition of a cryo-lyoprotectant can inducemicrostructural collapse of the dosage form matrix during freeze drying.As such, a balance should be struck to minimize microstructural collapseand at the same time preserving a sufficient number of virosomes tomaintain the virosome quality for inducing an immune response. Theamount of cryo-lyoprotectant in the base matrix formulation can be about0.01-2% w/w, about 0.1-1.5% w/w, about 0.2-1% w/w, or about 0.25-0.75%w/w. As such, the net amount (hereinafter “(net)”) of thecryo-lyoprotectant in the virosome formulation (i.e., liquid virosomeconcentrate plus base matrix formulation) can be about 0.5-6% w/w, about0.5-5% w/w, about 0.5-4.5% w/w, about 1-4.5% w/w, about 1.5-4.5% w/w,about 1.5-3% w/w, about 1.5-2.5, about 2-3% w/w, about 2.5% w/w, orabout 2% w/w. Applicants discovered that at these levels, thecyro/lyoprotectant can provide sufficient cryo-lyoprotection withoutresulting in unacceptable microstructural collapse during freeze drying.

In some embodiments, the base matrix formulation can also include amuco-adhesive such as gum. Suitable gums include, but are not limitedto, acacia, guar, agar, xanthan, gellan, carageenan, curdlan, konjac,locust bean, welan, gum tragacanth, gum arabic, gum karaya, gum ghatti,pectins, dextran, glucomannan, and alginates, or combinations thereof.

The base matrix formulation may also contain additional pharmaceuticallyacceptable agents or excipients. Such additional pharmaceuticallyacceptable agents or excipients include, without limitation, sugars,such as mannitol, dextrose, and lactose, inorganic salts, such as sodiumchloride and aluminum silicates, gelatins of mammalian origin, fishgelatin, modified starches, preservatives, antioxidants, surfactants,viscosity enhancers, permeability enhancers, coloring agents, flavoringagents, pH modifiers, sweeteners, taste-masking agents, and combinationsthereof. Suitable coloring agents can include red, black and yellow ironoxides and FD & C dyes such as FD & C Blue No. 2 and FD & C Red No. 40,and combinations thereof. Suitable flavoring agents can include mint,raspberry, licorice, orange, lemon, grapefruit, caramel, vanilla, cherryand grape flavors and combinations of these. Suitable pH modifiers caninclude citric acid, tartaric acid, phosphoric acid, hydrochloric acid,maleic acid, sodium hydroxide (e.g., 3% w/w sodium hydroxide solution),and combinations thereof. In some embodiments, the base matrixformulation and/or virosome formulation has an amount of a pH modifierto maintain a target pH of about 6-9, about 7-8, about 7.2-7.6, about7.3-7.5, or about 7.4. Suitable sweeteners can include aspartame,acesulfame K and thaumatin, and combinations thereof. One of ordinaryskill in the art can readily determine suitable amounts of these variousadditional excipients if desired.

The base matrix formulation can also include a solvent. In someembodiments, the solvent can be water (e.g., purified water). In someembodiments, the balance remaining of the base matrix formulation and/orvirosome formulation is the solvent.

The base matrix formulation can be about 25-99% w/w, about 35-90% w/w,about 40-85% w/w, about 45-80% w/w, or about 50-75% w/w of the virosomeformulation. In some embodiments, the base matrix formulation can beabout 65-85% w/w, about 70-80% w/w, about 73-77% w/w, or about 75% w/wof the virosome formulation.

Making a Dosage Form Comprising the Virosome Formulation

As stated above, a liquid virosome concentrate is mixed with a basematrix formulation to form a virosome formulation in step 101 suitablefor the freeze-drying process. FIG. 2 provides a more detaileddescription of the process of forming a vaccine dosage form disclosedherein. In some embodiments, the base matrix formulation can be preparedby dissolving a matrix former and a structure former in a solvent toform a premix. For example, gelatin and mannitol can be dissolved inwater as shown in step 201 of FIG. 2 . The premix can be heated to about40-80° C., about 50-70° C., about 55-65° C., or about 60° C. andmaintained for about 45-75 minutes, about 55-65 minutes, or about 60minutes. As shown in step 202, the premix can be heated to 60° C. andmaintained for 1 hour. The premix can then be cooled to about 30-50° C.,about 35-45° C., or about 40° C. and sieved before cooling down furtherto about 10-20° C. or about 15° C. and maintained at this temperaturethroughout the rest of the process. As shown in step 203, the premix canbe cooled to 40° C. and sieved. Next, the premix can be cooled to 15° C.as shown in step 204.

Next, the cryo-lyoprotectant can be added to the premix. For example,trehalose can be added to the premix as shown in step 205. Subsequently,the pH can be adjusted to about 6-9, about 7-8, about 7.2-7.6, about7.3-7.5, or about 7.4 using a pH modifier. For example, the pH can beadjusted to 7.4 using a sodium hydroxide solution as shown in step 206.After the pH is adjusted, the liquid virosome concentrate can be added.After the liquid virosome concentrate is added, the pH can be rechecked(step 207) and, if necessary, adjusted to about 6.0-8.5, about 7-8,about 7.2-7.6, about 7.3-7.5, or about 7.4 using additional pH modifier.This mixture can be made up to a desired batch size with solvent (i.e.,the virosome formulation). For example, an amount of water as necessarycan be added to the mixture as shown in step 208.

At step 102 of FIG. 1 , the liquid virosome formulation can be dosedinto a preformed mold. As used herein, “dosed” refers to the depositionof a pre-determined aliquot of solution or suspension. As used herein,“preformed mold” refers to any suitable container or compartment intowhich an aqueous solution or suspension may be deposited and withinwhich subsequently freeze dried. In certain embodiments of the presentdisclosure, the preformed mold is a blister pack with one or moreblister pockets. Predetermined aliquots in an amount of about 150-1000mg or about 500 mg wet filling dosing weight (also referred to as dosingfill weight) of the virosome formulation can be metered into preformedmolds. In some embodiments, the virosome formulation can be dosed atabout 10-20° C. or about 15° C. For example, the virosome formulationcan be dosed at 15° C. with a 500 mg dosing fill weight as shown in step209.

At step 103 of FIG. 1 , the dosed virosome formulations can then befrozen in the preformed molds. The dosed virosome formulations in thepreformed molds can be frozen by any means known in the art. Forexample, the formulations can be passed through a cryogenic chamber(e.g., liquid nitrogen tunnel). The temperature during freezing can bebetween about −50 to −100° C., about −60 to −90° C., about −60 to −80°C., about −65 to −75° C., or about −70° C. The freezing duration canrange from about 1.5-5 minutes, about 2-4.5 minutes, about 2.5-4minutes, about 3-4 minutes, about 3-3.5 minutes, or about 3.25 minutes.For example, the dosed virosome formulation can be frozen at −70° C. for3 mins and 15 seconds as shown in step 210.

At step 104 of FIG. 1 , the frozen units in the preformed molds can becollected and placed in a freezer at a temperature of less than about−25° C., about −20° C., about −15° C., about −10° C., about −5° C. andannealed (i.e., frozen hold) for a period of time to crystallize thestructure former. Structure former crystallization can provide thefrozen units with the structural strength to prevent the collapse of thefrozen units during freeze drying. This can be critical to the virosomeintegrity. The annealing time can range from about 3-9 hours, about 4-8hours, about 5-7 hours, or about 6 hours. For example, the frozen unitscan be annealed at less than −15° C. for about 3-9 hours as shown instep 211.

After annealing, the annealed frozen units can be freeze-dried in step105 to form the dosage form. During the freeze-drying process, the wateris sublimated from the frozen units. In some embodiments, the frozenunits can be loaded onto the shelves of a freeze-drier. In someembodiments, the freeze-drier can be precooled to about −15 to −35° C.,about −20 to −30° C., or about −25° C. Once the annealed frozen unitsare in the freeze-drier, the freeze-drying cycle can be initiated. Insome embodiments, a vacuum can be pulled and the shelf temperatureraised once the freeze-drying cycle is initiated. The freeze-drier canoperate at low pressure (i.e., vacuum). In some embodiments, thefreeze-drier can operate at a pressure of about less than or equal to1000 mbar, about 900 mbar, about 800 mbar, about 700 mbar, about 600mbar, about 500 mbar, or about 400 mbar.

Applicants discovered a two-step freeze-drying cycle (step 212) that canachieve structural robustness of the dosage form as well as minimallydamaging the virosome in the dosage form. The two-step freeze-dryingcycle can include a first step of holding the frozen units at about −5°C. to −25° C., about −10° C. to −20° C., about −13° C. to −17° C., orabout −15° C. for about 12-36 hours, about 18-30 hours, about 20-28hours, or about 24 hours. In addition, the two-step freeze-drying cyclecan include a second step that follows the first step. The second stepcan include holding the frozen units at about 0° C. to −20° C., about−5° C. to about −15° C., about −8° C. to about −12° C., or about −10° C.for about 6-30 hours, about 12-24 hours, about 14-22 hours, or about 18hours. At the end of the two-step freeze drying cycle, the temperatureof the freeze-drier can be raised to about ambient temperature (i.e.,about 20-25° C. or about 23° C.

In some embodiments, the two-stage freeze-drying process can includepre-cooling to the freeze-drier to about −25° C., ramping thefreeze-drier for 2 hours to −15° C., holding the freeze-drier at −15° C.for 24 hours, ramping the freeze-drier to −10° C. for 2 hours, holdingat −10° C. for 18 hours, ramping to 0° C. for 15 mins, and ramping to23° C. for 15 mins in that order.

The freeze-dried dosage forms can be removed from the freeze-drier andinspected for any defects (quality inspection as described below) atstep 213. The dosage forms can then be placed in a storage cabinet atatmospheric humidity less than about 35% RH before the dosage forms canbe sealed in their preformed molds. The sealing process (step 214) canplace a lidding foil on the preformed molds and provide blisters offreeze-dried dosage forms.

The water in the freeze-dried dosage forms can be removed viasublimation during freeze-drying. Accordingly, the remainder of thevirosome concentrate in the freeze-dried dosage form excluding thecryo-lyoprotectants (i.e., the virosomes, antigens, adjuvants, andbuffer system remaining from the freeze-dried virosome concentrate) canbe about 1-5 wt. %, about 2-4 wt. %, about 2.5-3.5 wt. %, about 2.6-3.4wt. %, about 2.7-3.3 wt. %, about 2.8-3.2 wt. %, about 2.9-3.1 wt. %, orabout 3-3.1 wt. % of the dosage form.

As stated above, the target molecule(s) is included in the dosage formsdisclosed herein in an amount, which is sufficient to render itimmunogenic when provided in a dosage form. A person of skill in the artcan readily determine the immunogenic amount for a given disease orinfection based on, among other facts, route of administration, age andweight of the patient to whom the dosage form will be administered. Insome embodiments, the solid dosage form can contain from 0.01-250 μg ofeach target molecule (e.g., HIV-1 P1 peptide and/or rgp41).

In some embodiments, at least one of the cryo-lyoprotectants in thefreeze-dried dosage form can be about 5-20 wt. %, about 8-18 wt. %,about 10-15 wt. %, about 11-15 wt. %, or about 12-15 wt. % of the dosageform. In some embodiments, at least one of the cryo-lyoprotectants inthe freeze-dried dosage form can be about 1-5 wt. %, about 1-4 wt. %, orabout 2-4 wt. % of the dosage form.

In some embodiments, the amount of matrix former in the dosage form canbe about 20-50 wt. %, about 25-45 wt. %, about 25-40 wt. %, about 30-40wt. %, about 33-37 wt. %, or about 35-37 wt. %. In some embodiments, theamount of structure former in the dosage form can be about 27-65 wt. %,about 27-60 wt. %, about 40-55 wt. %, or about 45-50 wt. %. In someembodiments, the remainder of the pH modifier in the freeze-dried dosage(e.g., sodium hydroxide) can be about 0.01-0.08 wt. %.

The dosage forms of the present disclosure are dissolving dosage formsand accordingly have the distinct advantage of a faster disintegratingtime. The route of administration may be oral, vaginal or nasal, thoughpreferably oral (i.e., sublingual and/or buccal). Once placed in theoral cavity and in contact with saliva, a dosage form can disintegratewithin about 1 to about 180 seconds, about 1 to about 120 seconds, about1 to about 60 seconds, preferably within about 1 to about 30 seconds,more preferably within about 1 to about 10 seconds and most preferablyin less than about 5 seconds.

Formulations, Test Methods, and Examples

For the examples, a liquid virosome concentrate prepared from influenzavirus was used. The virosomes contained the influenza HA as well asadded antigens and adjuvants. Two virosome preparations were made, eachcontaining a single antigen derived from the HIV envelope glycoprotein.The liquid virosome concentrate was a mixture of two virosomepreparations. The two HIV-gp41 derived antigens were the P1 peptiderepresenting the last 35 C-terminal ectodomain residues and thetruncated rgp41 devoid of cluster I and the last 21 C-terminalectodomain residue. Adjuvant 3M-052 was either present or absent ineither virosome preparation. The virosomes were suspended inHEPES-Sodium Chloride buffer containing 142.5 mM sodium chloride and 50mM HEPES at pH 7.4. In addition, trehalose (a cryo-lyoprotectant) wastested in the range of 0-10% w/w of the liquid virosome concentrate. Thefollowing Table 1 summarizes the target compositions of the HIV-1 liquidvirosome concentrate used for some of our experiments, during which analiquot of 500 mg (dosing fill weight) of the aqueous virosomeformulation was metered into pockets preformed blister, followed byfreezing and freeze drying. Dosing fill weights may range from 150 mg to1000 mg and the compositions of the HIV-1 liquid virosome concentratecan be adjusted to meet the target dose required. Please note that thefollowing target ranges can be dependent on the purpose and the furtheruse, e.g., for animal studies or for human studies. Thus, the othertarget concentrations may be useful for other purposes.

TABLE 1 Example of Lipid based Particle Concentrates for a 500 mg dosingfill Example Target Molecules and Excipients weight Zydis ® virosomeformulation In Lipid based Particle Concentrates HIV vaccine MYM-V202Target molecule (P1 antigen): 50-450 μg/mL (4x concentrate for 25%loading Target molecule (rgp41 antigen): 50-400 μg/mL of liquid virosomein the Zydis ® HA excipient: 10-160 μg/mL virosome formulation Adjuvant(e.g. 3 M-052): 8-140 μg/ml Phospholipids: 0.5 to 5 mg/mL SodiumChloride: 50-150 mM HEPES 10-50 mM Trehalose: 4-10% w/w pH 6.5 to 8.0HIV vaccine MYM-V202 Target molecule (P1 antigen): 25-225 μg/mL (2xconcentrate for 50% loading Target molecule (rgp41 antigen): 25-200μg/mL of liquid virosome in the Zydis ® HA excipient: 5-80 μg/mLvirosome formulation) Adjuvant (e.g. 3 M-052): 4-70 μg/ml Phospholipids:0.5 to 5 mg/mL Sodium Chloride: 50-150 mM HEPES 10-50 mM Trehalose: 2-5%w/w pH 6.5 to 8.0 Placebo vaccine VP02 HA excipient: 10-160 μg/mL (4xconcentrate for 25% loading Adjuvant (e.g. 3 M-052): 8-140 μg/ml ofliquid virosome in final Zydis ® Phospholipids: 0.5 to 5 mg/mL virosomeformulation) Sodium Chloride: 50-150 mM HEPES 10-50 mM Trehalose: 4-10%w/w pH 6.5 to 8.0

In some of our experiments, the target dose for HA and the HIV-1antigens for each tablet were 20 μg HA, 25 μg P1, and 50 μg rgp41. Toachieve these doses, a high payload of the liquid virosome concentratesin combination with high wet filling dose weight were required. Table 2shows the various combinations of liquid virosome concentrate loading(ranging from 25-50% w/w) with wet fill dosing weight (ranging from 250to 1000 mg). The wet fill dosing wet is the amount of aliquot of thevirosome formulation metered per dose prior to freeze-drying.

TABLE 2 Liquid virosome Virosome vaccine Liquid virosome concentrateTarget dose concentrate HA % loading in base Dosing fill per tablet andantigen content matrix formulation weight HA/P1/rpg41 HA 80 μg/ml (with25% w/w 1000 mg 20 μg/25 μg/50 μg adjuvant 3 M -052) 50% w/w 500 mg 20μg/25 μg/50 μg P1 100 μl/ml rgp41 200 μg/ml HA 160 μg/ml (with 25% w/w500 mg 20 μg/25 μg/50 μg adjuvant 3 M -052) 50% w/w 250 mg 20 μg/25μg/50 μg P1 200 μl/ml rgp41 400 μg/ml

A 25% loading of the liquid virosome concentrate can be added to thebase matrix formulation. The dosing filling dose weight can be 500 mg.Table 3 shows the range of HA and antigen contents evaluated.

TABLE 3 Target Composition Composition in composition in Supplied for a25% loading Component Liquid concentrate evaluation formulation VirosomeHA: 10-160 μg/ml HA: 70-160 μg/ml HA: 18-40 μg/ml tagged with P1: 50-450μg/ml P1: 40-100 μg/ml P1: 10-25 μg/ml antigens and rgp41: 50-400 μg/mlrgp41: 70-230 μg/ml rgp41: 17.5-57.5 μg/ml adjuvants 3 M-052: 8-140μg/ml 3 M-052: 16-65 μg/ml 3 M-052: 4-16.3 μg/ml Sodium 50-150 mM 142.5mM 35.625 mM Chloride HEPES 10-50 mM 50 mM 12.5 mM Trehalose 4-10% w/w3.5-7% w/w 0.9-1.75% w/w

The presence of buffer in the aqueous composition can depress thefreezing point, thus making it difficult to freeze the formulationcomposition and maintain its frozen state. In addition, collapse of thetablet matrix structure can also occur during the freeze-drying asbuffer salts can depress the crystallization of mannitol during theanneal process. Crystallization of mannitol is required to providestrength and structure to the tablet matrix to prevent structurecollapse. However, crystallization of mannitol can damage the virosomeparticles during freezing, annealing, and freeze-drying. A lowerpercentage loading of the liquid virosome concentrate (e.g. 25% loading)lands to reducing this impact. A combination of lower percentage loadingof the liquid virosome concentrate and a larger dosing fill weight canalso be considered.

A high wet fill dosing weight of the virosome formulation in combinationwith a formulation composition with high buffer content can also make itmore difficult to freeze and maintain the structure to minimize collapseduring freeze-drying. However larger tablets (e.g. 1000 mg dosing fillweight) can cover bigger surfaces area and can potentially improvevirosome passage. When a high wet fill dosing weight is required,formulation composition with low buffer content is preferred.

Table 4 (Formulation 1) summarizes the aqueous compositions of thevirosome formulation and the corresponding composition for the tabletvirosome evaluated herein. The following formulations and tablets weremade according to the steps shown and described in FIG. 2 . In addition,the frozen formulations were subjected to the two-step freeze-dryingprocess with a vacuum of 500 mbar of: (a) −25° C. pre-cool; (b) Ramp for2 hours to −15° C.; (c) Hold @ −15° C. for 24 hours; (d) Ramp to −10° C.for 2 hours; (e) Hold @ −10° C. for 18 hours; (f) Ramp for 15 mins to 0°C.; (g) Ramp for 15 mins to 23° C. Next, the vacuum was released and thefreeze drier returned to atmospheric pressure.

The concentration of each ingredient (% w/w) is the amount prior toremoval of the water present in the liquid virosome concentrate, sodiumhydroxide solution, and the water used for preparing by sublimationduring lyophilization. Also, the following table includes amounts whicheach ingredient may be ranged.

TABLE 4 Amount in mg Amount in mg % w/w for a 500 mg in post % w/w rangeFormulation wet dosing fill freeze Ingredient evaluated 1/2 weightdrying Liquid Virosome 25-50% 25% 125 mg ~2.5-2.6 mg* concentrateTrehalose 0.5%-4.5%  2%** (net) 10 mg 10 mg** (net) Fish Gelatin 4-6% 6% 30 mg 30 mg Mannitol 4.5-8%  4.5%/8% 40 mg 40 mg Sodium Hydroxide qspH 7.4 qs pH 7.4 ~1.3 mg (qs ~0.04 mg (water Solution (e.g. 3% pH 7.4)removed by w/w) sublimation) Water (for preparing qs 100% qs 100% ~293.7mg Water the aqueous matrix removed mixture) Total weight of N/A N/A N/A~82.64 mg freeze dried vaccine tablet *As stated above, the liquidvirosome concentrate included virosomes suspended in a buffer system of142.5 mM NaCl and 50 mM HEPES with 5% w/w trehalose. For a 25% loadingof the virosome formulation in a 500 mg dosing fill weight (i.e., 125 mgof the liquid virosome concentrate), the estimate dry matter with watersublimated of the virosomes, antigens, adjuvants, NaCl, and HEPES(excluding trehalose) is about 2.5-2.6 mg. **This is the net amount oftrehalose in the virosome formulation and the dosage form. As such, thisamount includes trehalose from the liquid virosome concentrate as wellas trehalose added from the base matrix formulation.Properties of Freeze-Dried Dosage Forms:

The freeze-dried dosage forms can be stable in physical attributes andvirosome quality (particle size characteristics and antigen content) andcan be stored independent of cold chain storage conditions. In addition,the freeze-dried dosage forms can allow the virosomes to be resistant toaccidental exposure to sub-zero storage conditions during storage ortransportation.

Dosage Form Physical Characteristics:

Freeze dried tablets with acceptable physical characteristics wereproduced. The physical attributes of the tablets include appearance,dispersion characteristics, disintegration times, and moisture content.

Tablet Appearance of ten freeze-dried tablets are tested. Each tablet isremoved from the blister package. A visual inspection on each tablet forsurface defect on the tablet surface and base is performed. Thecriterion is that the freeze-dried tablet should have good appearancewith no surface defects. In addition, the tablets should be ofsufficient robustness for their removability from the blister pocketwithout breakage.

Dispersion Characteristics (in-vitro test): A minimum of 5 tablets aretested. First, a beaker is prepared containing approximately 200 mL ofpurified water at 20° C.±0.5° C. Each tablet is then removed from theblister package and the tablet is placed base down on the surface of thewater. The time is taken for the time each tablet takes to fully wet ordissociate. Wetting the time taken for the unit to fully wet. Thewetting of the tablet can occur in patches, eventually merging togetherso that the whole unit is wet. The dispersion test is consideredcomplete when the center of the unit is a wetted mass. Thus, the wettingtime is taken from when the center of the unit has wetted through asthis is the thickest part of the unit. The wetting time is recorded foreach of the five tablets. The maximum time for each test is 60 seconds.Time longer than this may be written simply as greater than 60 seconds.Dissociation=the time taken for the unit to break apart. This time canbe taken when the unit starts to fall apart at the edges. Thedissociation time is recorded for each of the five tablets. The maximumtime for each test is 60 seconds. Times longer than this may be writtenas greater than 60 seconds. Occasionally, the unit will not fully wet ordissociate completely inside of this time limit. At times, the unit mayhave hard lumps in it; other times it may have not wetted on the surfaceat all. In addition, the whole unit may be covered in a hard skin. Anote of this is made in the description if it happens, citing “hardlumps”, or “skin remains”, as appropriate. The formation of “hard lumps”and/or “skin” can be an indication of microstructural collapse duringfreeze-drying. FIGS. 3A-C show a simplified representation of the threepossible non-dispersed states, with a side view and a top view of theunits as they would appear in the water. The photos in FIGS. 3A-C showsome representative units for the same categories. The criterion for thedispersion characteristic test is if the 5 tablets can be fully wettedand/or dissociated into a palpable mass without the presence of hardlumps and skin in 60 seconds or less. In some embodiments, the dosageforms disclosed herein can be fully wetted and/or dissociated into apalpable mass without the presence of hard lumps and/or skin in 60seconds or less.

Disintegration Time (in-vitro test): Six tablets are used for this test.Six beakers are filled with purified water and placed in a water bathcontrolled at 37° C.±0.5° C. Each tablet is then removed from theblister package. Carefully place a wire clip onto each of the sixtablets. Ensure that the clip grips the tablet without causing damage.Next, perform the test as described in the Pharmacopeia. An example ofsuch a test is the United State Pharmacopeia (701) Disintegration. Themaximum disintegration time is recorded for each tablet. The criterionfor the disintegration time test is that the disintegration time shouldnot be more than 60 seconds for each of the six tablets. In someembodiments, the dosage forms disclosed herein can have a disintegrationtime of less than 60 seconds.

Moisture Content: A Methron 831 Karl Fischer Coulometer with a 744 OvenSample Processor (Metrohm, Herisau, Switzerland) is used to determinethe water content of the tablet. A tablet is accurately weighed andplaced it in a glass vial. The vial is crimp shut immediately to ensureno moisture ingress. Then the sample vial is placed in the 744 OvenSample Processor and set the temperature to 102° C. The evaporatedmoisture is titrated using a Hydranal Coulometric AG Oven reagent toquantify the amount of water released. The test is performed intriplicate and the mean is recorded. The criterion for the moisturecontent is if the freeze-dried dosage form has a moisture content ofless than about 8%, preferably less than 6%, and more preferably lessthan 4%. In some embodiments, the dosage forms disclosed herein can havea moisture content of less than about 8%, preferably less than 6%, andmore preferably less than 4%.

Virosome Characteristics:

The freeze-dried dosage forms that contain the virosomes can besufficiently preserved in terms of proportion of intact virosomes fromthe starting liquid virosome population, its particle size and surfaceantigens content required for the immunogenicity and immunologicalbenefits of the virosomes. The virosome structure can be destroyed bythe freeze-drying process used for producing the dosage forms. As such,virosome particulate characteristics are assessed from a solution of areconstituted freeze-dried tablet. The virosomes can exist as intactindividual particles and as clusters of different sizes, all beingimmunogenic but having different antigen/epitope exposure, and harboringdifferent ability to cross the sublingual barrier. The virosomes may becharacterized in terms of (a) mean particle size and (b) proportion ofvirosome preservation (intact virosomes).

Virosome Particle Size and Particle Concentration (counts of virosomeparticles per mL) assessment using NTA technique: Nanoparticle TrackingAnalysis (NTA; with NanoSight NS300 instrument) is a sensitive methodthat can identify different particle sizes in the range of 30-1000 nmpresent in a solution. Particles in a sample solution can individuallybe tracked and simultaneously analyzed by direct observation.Nanoparticles move under Brownian movement due to the random movement ofwater molecules surrounding them. Small particles move faster thanlarger particles. Brownian motion of each particle is followed inreal-time via video and the NTA analyzes the Brownian motion todetermine the particle size. The diffusion coefficient can be calculatedby tracking the movement of each particle and then through theapplication of the Stokes-Einstein equation, the particle size can becalculated. This particle-by-particle methodology produces highresolution results for particle size distribution and concentration(i.e., number of particles in a known volume of liquid). The targetvalues for optimum detection for the instrument detection are summarizedin the following Table 5.

TABLE 5 Quality Indicator Target Rationale PPF 50-100 Particle countaffects (Particles Per Frame) resolution achievable and statisticalaccuracy of profiles generated Number of Valid Tracks >5000 Statisticalaccuracy of profiles generated for poly-disperse samples S:N (Signal toNoise High Improved detection of ratio) small or faint particles SD(Standard Deviation) Low Less variable data gives higher confidence inresults Appearance of profile Good: non- Less noise present in data,Gaussian, clear particle size minimal populations shouldering on peaks

The test sample must be in liquid form. For this work, the virosomevaccine is supplied in liquid form. For test samples from the vaccineformulations disclosed herein, the mix is supplied in liquid form, afrozen unit is thawed to liquid form before testing, and a freeze-drieddosage form is reconstituted with water buffer to liquid form. The testsample should not be too concentrated. A liquid test sample can bediluted further with HN buffer as appropriate to optimize the detection.The dilution buffer can be of high purity, and can be filtered at leastthrough a 0.22 μm filter prior to use. The experimental parameters setfor NTA analysis are summarized in the following Table 6.

TABLE 6 Parameter Setting Sample Dilution Dependent on content (canrange from 1:100 to 1:8000 depending on the concentration of virosomecounts in the diluted test samples) Number of Captures 5 CaptureDuration 60 seconds Temperature control 25° C. Viscosity 0.9 Cp

The samples were measured by NTA as known in the art. In someembodiments, the virosome particle range including fragments andclusters disclosed herein can be from about 50-500 nm. In someembodiments the virosomes particles that are intact virosomes can be inthe range of about 70-400 nm or about 70-200 nm (main peak). In someembodiments, the mean diameter of the virosome particles can be about70-200 nm, about 100-175 nm, about 125-155 nm. For detection purpose,the particle concentration of virosome population in the sample can beat least 10¹⁰ counts/mL.

Proportions of Virosome Preservation Assessment Using Flow Cytometry:Flow cytometry is used for this assessment. First, the starting liquidvirosome particles is labelled (reference sample representing 100% ofthe starting material) by inserting a lipophilic dye Dil (long-chaindialylcarbocyanin) into the lipid bilayer of the virosomes. (Labellingwith Dil has no measurable effect on the particle size). Secondly, thefreeze-dried tablet is reconstituted and labeled the virosomes in thefreeze-dried tablet with Dil (the test sample). Then the samples areanalyzed using AMNIS imaging flow cytometer and the events between thereference (liquid virosomes before freeze-drying) and the test samples(freeze-dried virosomes) are compared to estimate the proportions ofvirosome preservation following freeze drying in terms of: (a)percentage of virosome recovery and (b) percentage of virosome clusters.In some embodiments, the percentage of recovery of the virosome assingle particles can be about 20-50%, about 30-50%, or about 40-50% ofthe starting material. In some embodiments, the percentage of virosomeclusters (doublets, triplets or higher number forms) can be about lessthan 50%, about 25%, about 10%, or about 5%.

Content of Virosome Hemagglutinin (HA), HIV-1 Antigen P1, HIV-1 Antigensand Adjuvant

The influenza HA, HIV-1 antigens, and adjuvant contents of the virosomecan be quantified by various methods. These are tabulated in Table 7below.

TABLE 7 Content to be quantified Methods Influenza HA Immunoblot assaySRID assay ELISA HPLC assay HIV-1 antigens Immunoblot assay HPLC assayELISA Adjuvant UV spectroscopy assay HPLC assay

Immunoblot Assay Method for HA, P1, and rgp41: In this analysis, theformulation (liquid virosome concentrates or reconstituted freeze-drieddosage forms containing the virosomes) can be absorbed ontonitrocellulose membrane and the antigens are maintained in their nativestate due to the absence of heating procedure, denaturing or reducingagents. This assay detects all antigens accessible to the specificantibodies and is indicative for major antigen degradation ordenaturation that destroys or blocks access to the specific epitope. Italso indicates if specific excipients could prevent antibody binding toits antigen. To prepare the freeze-dried dosage forms for analysis (testsamples), each tablet is dissolved in 0.5 mL of water (i.e.,reconstitute the freeze-dried tablet back to the composition of virosomeformulation prior to freeze-drying). To prepare a positive control, asample of liquid virosome concentrate is diluted with ultrapure water(the dilution is 4 folds). The liquid virosome concentrate ideally isthe same batch that is used in making the dosage form test sample.Serial 2-fold dilutions of all the test samples and the positive controlare prepared. Additional positive controls like purified HA and rgp41,and synthetic P1 may be used as well. A 1.5 μL of each sample and thepositive control is spotted onto the dry nitrocellulose membrane (withincreasing dilution from left to right—undiluted, ½, ¼, ⅛, 1/16, 1/32,1/64, 1/128). The dry membrane is blocked with 1% (w/v) casein andincubate with specific antibody solution—the human monoclonal antibody(mab) 2F5 specific for HIV-1 P1 antigen and the rabbit anti-rgp41 serumfor HIV-1 rgp41 antigen. After incubation, the nitrocellulose membraneis washed. Then, the bound specific antibodies with fluorescent labelledsecondary antibodies (anti-human or anti-rabbit) are detected using afar-red fluorescence scanner that allows simultaneous detection of 2different fluorescent labels at 700 nm and 800 nm. The fluorescence rawdata signal for each sample spot (from lowest to highest) is compared tothe respective dilution of the positive control spot (in % of thepositive control). An arithmetic average of the sample percentage iscalculated.

UV Spectroscopy Assay for 3M-052: The adjuvant molecule 3M-052 hasseveral UV adsorption peaks. Some of these peaks overlap with theabsorption of lipids and proteins. Determination was done by UVspectroscopy at 320 nm.

HPLC Assay for P1 and rgp41: The HIV antigens P1 and rgp41 wereseparated by reversed phase high pressure liquid chromatography on a C18column using a water to acetonitrile gradient. Determination was done byUV spectroscopy at 280 nm, and peak areas were quantified by the HPLCsystem software.

ELISA endpoint antibody titer: Maxisorp 96-well plate (Nunc-flat bottom)and Polysorp plates were respectively coated at 4° C. for 16 hours with0.1 mL of rgp41 or P1 peptide (2 μg/mL) prepared in PBS pH 7.4. Plateswere washed 3 times with PBS with 0.05% v/v) TWEEN® 20 (PBST), then theblocking solution 1% BSA prepared in PBST was added to each well andincubated 2 hours at room temperature (RT). Plates were washed threetimes with PBST prior adding 0.1 mL per well of pre-immune serum dilutedat 1/1000 or immune serum serial dilutions (from 1/1000 to 1/64,000)prepared in 0.1% BSA in PBST and incubated for 2 hours at RT. Plateswere washed three times with PBST and incubated for 2 hours at RT withthe goat anti-rat IgG-HRP diluted 1:4000 in 0.1% BSA in PBST. Plateswere washed again before adding 0.1 mL of the colorimetric substrateo-phenylenediamine (OPD) and the reaction was stopped with 2M H2504,followed by plate reading at 492 nm.

Example 1: Use of High Mannitol Level (8% w/w) in Conjunction with LowTemperature Freeze Drying Cycle to Reduce Microstructural CollapseDuring Freeze Drying without Damaging the Virosome Integrity

Mannitol is used in dosage forms to increase structural robustness. Dueto the presence of high levels of buffers and the addition of trehaloseto protect the virosome particles, the use of mannitol at typical levelof 4.5% w/w was unable to provide sufficient structural support duringfreeze drying. Thus, microstructural collapse occurred.

However, Applicants discovered that by using a combination of higherlevel of mannitol and low temperature freeze drying cycles, astructurally more robust freeze-dried tablet can be achieved. Thisexample shows data comparing a formulation containing 4.5% w/w mannitolwith that containing 8% w/w mannitol. In both formulations, a 25% w/wloading of liquid virosome concentrates MYM-201 lot 160125-1 supplied byMymetics containing HA 70-80 μg/ml, P1 40-50 μg/ml, rgp41 70-80 μg/ml(A/Brisbane/59/2007 (H1N1) in HN buffer pH 7.4 (50 mM HEPES, 142.5 mMNaCl) was used in the virosome formulation. The fish gelatine and nettrehalose levels were kept at 6% w/w and 2% w/w of the virosomeformulation, respectively. As illustrated in FIG. 2 , the formulationswere dosed with a 500 mg dosing fill weight aliquot at 15° C. intoblister pockets of aluminum trays. The trays containing the dosedaqueous vaccine mix were frozen by passing the aluminium trays through afreezing chamber set at −70° C. for a duration of 3 minutes 15 seconds.The aluminium trays containing the frozen products were collected andplaced in a freezer at a temperature of <−15° C. and annealed for 6hours frozen hold before lyophilisation. A 2-step FD cycle using −15° C.for 24 hours followed by −10° C. for 18 hours was then used. Theblisters of freeze-dried tablets were sealed in sachets and stored atambient condition.

The physical characterisation of the lyophilised tablets (appearance anddispersion time) were assessed according to the tests explained above.In addition, the virosome particle size in the lyophilised tablets werecharacterised according to the tests explained above. The results aresummarised in Table 8 below.

TABLE 8 Mannitol 4.5% (Formulation 1) Mannitol 8% (Formulation 2)Appearance: Good (n = 182) Appearance: Good (n = 184) Dispersion Time (n= 8) Dispersion Time (n = 8) Wetting: >60 s (2 units-skin, 5 units-Wetting: <28 s hard lumps Dissociation: <36 s Dissociation: >60 s(didn't fully NTA Analysis dispersed) Freeze dried tablet: 140 nm (mainpeak) NTA Analysis % virosome particle (<200 nm): >50% Freeze driedtablet: 136 nm (main peak) % virosome particle (<200 nm): >50%

All the tablets had good appearance. The 4.5% w/w mannitol formulationhas poor dispersion behaviour with skin and/or hard lumps seen in thetablets due to microstructural collapse. Increasing the formulation to8% w/w which promotes the crystallisation of mannitol improved structureof the freeze-dried tablet. The formulation with 8% w/w mannitol has agood dispersion behaviour and gave a soft and palpable mass. Incombination with low temperature freeze drying cycle, the virosomeparticle in the freeze-dried tablet was not affected. The virosomeparticle size in the freeze-dried tablets was comparable overall betweenthe formulations.

Example 2: Stability Data of Freeze-Dried Vaccine Dosage Forms(Formulation 1) Stored for 3 Months Under ICH Conditions

The liquid virosome concentrates is generally stable at 2-8° C. but theliquid HIV virosomal vaccine has a limited shelf life of few months dueto important chemical modifications taking place on the antigens, whilethere is no aggregation. With solid vaccine form with low moisturecontent, such modifications are expected to be slowed down and minimalovertime, extending the shelf life >1 year. The stability of thevirosome vaccine in the form of a freeze-dried tablet Formulation 1 isillustrated in this example. Liquid virosome concentrate batch MYM V202lot 170130-1 supplied by Mymetics comprising of approximately 100 μg/mlHIV-1 P1, 230 μg/ml HIV-1 rgp41, 130 μg/ml HA (A/Brisbane/59/2007(H1N1), 65 μg/ml adjuvant 3M-052 in HN buffer pH 7.4 (50 mM HEPES, 142.5mM NaCl) with 7% w/w trehalose was supplied for the manufacture oflyophilized vaccine tablets (batch Z33787A101).

To prepare the vaccine tablet, a liquid virosome formulation mix wasprepared first. It contained 25% w/w of liquid virosome concentratebatch MYM V202 lot 170130-1, 6% w/w fish gelatin, 4.5% w/w mannitol, 2%w/w (net) trehalose, sodium hydroxide solution (quantum satis) to pH7.4, and purified water (quantum satis) to 100% w/w.

The liquid virosome formulation mixture was dosed with a 500 mg dosingfill weight aliquot at 15° C. into blister pockets of aluminum trays.The trays containing the dosed aqueous vaccine mix were frozen bypassing the trays through a freezing chamber set at −70° C. for aduration of 3 minutes 15 seconds. The aluminium trays containing thefrozen products were collected and placed in a freezer at a temperatureof <−15° C. A 2-step FD cycle using −15° C. for 24 hours followed by−10° C. for 18 hours was then used.

The blisters of freeze-dried tablets were sealed in sachets and placedon storage for 3 months at 5° C., 25° C./60% relative humidity, and 40°C./75 relative humidity. The results at initial testing and at 3 monthstesting are summarized in Table 9 below.

TABLE 9 25° C. @ 40° C. @ 5° C./ 60% RH/ 75% RH/ Test Initial 3 month 3month 3 month Appearance Good Good Good Good Moisture Content 5.22 5.205.76 4.83 (% w/w) Disintegration <13 <23 <41 <25 Time (sec) NTA: MainPeak 101 120 124 124 (nm) NTA: % virosome >50% >50% >50% >50% particle(<200 nm):

The stability data shows that the tablet appearance was good andconsistent between batches. The moisture content was 5-6% w/w withlittle difference between the different stability conditions over the3-month storage period. As this formulation contains 4.5% w/w mannitol,there is some microstructural collapse during freeze drying as indicatedin the variability in disintegration time. In terms of virosome particlesize, the DLS and NTA data showed no discernible difference in thevirosome particle size between tablets stored at the various stabilityconditions. The starting liquid virosome concentrates has >50% of theparticle <200 nm. The results show that virosome <200 nm from thereconstituted unit is essentially preserved to the similar order ofmagnitude.

Using semi-quantitative immunoblot analyses, antigens HIV-1 antigen P1and rgp41 were monitored for degradations over 3 months. All sampleswere pre-diluted 2-fold. The human mAb 2F5 specific for P1 and therabbit anti-rgp41 serum were used for this purpose. The liquid virosomeconcentrate batch MYM-V202 lot 170130-1 that was used to produce thevaccine batch was diluted 8-fold and used as a positive control. FIG. 4shows a photo of the immunoblot analysis. For sample Z33787A101, tabletsstored at 5° C. showed no or only a minimal decrease in the rgp41antigen signal after 1 month and 3 months of storage compared to theinitial sample (t=0). Similarly, there was only a minimal difference inthe signal intensity for the unit stored at 25° C. for the rgp41antigen, and a slight decrease for the P1 antigen. The difference forthe 40° C. units was more pronounced, although this might still bewithin the assay accuracy of 10-20%.

The quantitative evaluation for the fluorescence data for rgp41 and P1are presented in Tables 10 and 11, respectively. The upper part of eachtable shows the fluorescence raw data signal for each spot (from lowestto highest dilution) for the indicated stability sample. The lower partof each table shows the value for each sample spot compared to therespective dilution of the positive control spot (in % of the positivecontrol). The bottom line shows the arithmetic average of all samplepercentages. Obvious outliners of the measurements were excluded fromthe average calculation.

TABLE 10 Z33787A101 t = 0 1 month 3 months MYM-V202 5° C. 5° C. 25° C.40° C. 5° C. 25° C. 40° C. rgp41 7702462 9058972 8032375 9201721 78013446321957 10300125 8521558 4339215 5472388 4583281 4902260 4771430 40164244072924 3454675 5371152 4467722 3785349 3403051 4176710 3596774 25560363084636 3319677 3265426 2311220 3464764 3194101 3263683 3054427 26150032259183 2179005 2166937 2708839 2159053 2482939 2704366 2043011 17045341689938 1182171 1769144 1533427 1448772 1594263 1077611 1339732 1048682751850 1056018 675617 785497 976990 621839 679095 632157 517094 514737455636 490586 451385 417028 % of control 117.6 104.3 119.5 101.3 82.1133.7 110.6 % of control 126.1 107.9 113.0 110.0 92.6 93.9 79.6 % ofcontrol 83.2 70.5 63.4 77.8 67.0 47.6 57.4 % of control 98.4 69.6 104.496.2 98.3 92.0 78.8 % of control 96.5 95.9 119.9 95.6 109.9 119.7 90.4 %of control 99.1 69.4 103.8 90.0 85.0 93.5 63.2 % of control 78.3 56.178.8 50.4 58.6 72.9 46.4 % of control 93.1 76.1 75.8 67.1 72.2 66.5 61.4% (average) 99.0 81.2 97.3 86.0 74.0 80.0 65.3

TABLE 11 Z33787A101 t = 0 1 month 3 months MVM-V202 5° C. 5° C. 25° C.40° C. 5° C. 25° C. 40° C. P1 2764625 1461965 1245932 1471586 12385371516476 1351413 1572627 1208918 1606470 723609 1235559 1338350 547105470287 508107 845363 997068 578732 617270 997966 619197 352482 610820453354 904843 566560 879843 1200198 1211985 882260 490097 249520 317945287210 840751 673118 324793 339128 261055 149361 269482 142741 311659342849 146881 159835 115068 86167 110800 63348 127920 163997 12401091144 49784 28407 42578 19915 18230 19079 24436 27082 27289 % of control52.9 45.1 53.2 44.8 54.9 48.9 56.9 % of control 132.9 59.9 102.2 110.745.3 38.9 42.0 % of control 117.9 68.5 73.0 118.1 73.2 41.7 72.3 % ofcontrol 199.6 125.0 194.1 264.7 267.3 194.6 108.1 % of control 127.4115.1 336.9 269.8 130.2 135.9 104.6 % of control 180.4 95.6 208.7 229.598.3 107.0 77.0 % of control 128.6 73.5 148.5 190.3 143.9 105.8 57.8 %of control 149.9 70.1 64.2 67.2 86.0 95.3 96.1 % (average) 136.2 81.6147.6 161.9 112.4 96.0 76.8

For vaccine tablet for Z33787A101, tablets stored at 5° C. showed no oronly a minimal decrease in the rgp41 and P1 antigens signal after 1month and 3 months of storage compared to the initial sample (t=0).Similarly, there was only a minimal difference in the signal intensityfor the unit stored at 25° C. for the rgp41 antigen, and a slightdecrease for the P1 antigen. The difference for the 40° C. units wasmore pronounced for both antigens P1 and rpg41, although this mightstill be within the accuracy of the assay, especially since variationswere seen within the serial dilution samples again.

Determination of 3M-052 was done by UV spectroscopy at 320 nm for allvaccine tablets stored at different temperatures of over 3 months.Values are presented in Tables 12 below.

TABLE 12 Time point Storage condition OD (320 nm) 0 5° C. 0.116 1 month5° C. 0.114 1 month 25° C./60% RH 0.112 1 month 40° C./75% RH 0.110

No discernible difference can be observed between these samples andtherefore, 3M-052 concentrations were considered to remain stable in allsamples within the accuracy of this assay. Overall, HA, P1, rpg41 and3M-052 were stable in the freeze-dried tablets stored under differentstorage conditions, with minor variations over time as shown in Table 13below.

TABLE 13 Antigen and adjuvant detection during Liquid virosome stabilitystudy concentrates Freeze-dried tablets Changes in rgp41 over 3 monthsat 5° C. Not significant Not significant (degradation not expected =reference) Changes in rgp41 over 3 months at 25° C. Not done Minimaldecrease (degradation), as compared to 5° C. Changes in rgp41 over 3months at 37-40° C. Not done Minimal decrease (degradation), as comparedto 5° C. Changes in P1 over 3 months at 5° C. Not significant Notsignificant (degradation not expected = reference) Changes in P1 over 3months at 25° C. Not done Minimal decrease (degradation), as compared to5° C. Changes in P1 over 3 months at 37-40° C. Not done Minimal decrease(degradation), as compared to 5° C. Changes in 3 M-052 over 3 months at5° C. Not significant Not significant (degradation not expected =reference) Changes in 3 M-052 over 3 months at 37-40° C. Not significantNot significant (degradation), as compared to 5° C.

Example 3: Stability Data of Freeze-Dried Vaccine Dosage Forms(Formulation 2) Stored Under ICH Conditions

The stability of the virosome vaccine in the form of a freeze-driedtablet for Formulation 2 is illustrated in this example. Liquid virosomeconcentrate batch MYM V202 lot 17MYM002/F17255 supplied by Mymeticscomprising of approximately 121 μg/ml HIV-1 P1, 67 μg/ml HIV-1 rgp41, 41μg/ml HA (A/Brisbane/59/2007 (H1N1), 39 μg/ml adjuvant 3M-052 in HNbuffer pH 7.4 (50 mM HEPES, 142.5 mM NaCl) with 7% w/w trehalose wasused for the manufacture of lyophilized vaccine tablets (batch MYM-212lot 1690747).

To prepare the vaccine tablet, a liquid virosome formulation mix wasprepared first. It contained 25% w/w of liquid virosome concentratebatch MYM V202 lot 17MYM002/F17255, 6% w/w fish gelatin, 8% w/wmannitol, 2% w/w (net) trehalose, sodium hydroxide solution (quantumsatis) to pH 7.4, and purified water (quantum satis) to 100% w/w.

The liquid virosome formulation mixture was dosed with a 500 mg dosingfill weight aliquot at 15° C. into blister pockets of aluminum trays.The trays containing the dosed aqueous vaccine mix were frozen bypassing the trays through a freezing chamber set at −70° C. for aduration of 3 minutes 15 seconds. The aluminium trays containing thefrozen products were collected and placed in a freezer at a temperatureof <−15° C. A 2-step FD cycle using −15° C. for 24 hours followed by−10° C. for 18 hours was then used.

The blisters of freeze-dried tablets were sealed in sachets and placedon storage for 3 months at 5° C., 25° C./60% relative humidity, and 40°C./75 relative humidity. The results at initial testing and at 6 monthstesting are summarized in Table 14 below.

TABLE 14 Test Appearance Disintegration Time Moisture Content InitialGood 22 seconds 3.6% 1 month at 5° C. Good 8 seconds 3.8% 1 month at 25°C./60% RH Good 16 seconds 3.7% 1 month at 40° C./75% RH Good 14 seconds3.6% 3 months at 5° C. Good 12 seconds 3.7% 3 months at 25° C./60% RHGood 10 seconds 3.7% 3 months at 40° C./75% RH Good 13 seconds 3.9% 6months at 5° C. Good 8 seconds 3.9% 6 months at 25° C./60% RH Good 10seconds 3.8% 6 months at 40° C./75% RH Good 13 seconds 4.0%

The stability data shows that the tablet appearance was good andconsistent between batches. The moisture content was 3.6-4.0% w/w withlittle difference between the different stability conditions over the6-month storage period. As this formulation contains 8% w/w mannitol,there is less microstructural collapse during freeze drying as indicatedin the shorter and more consistent disintegration times.

The stability of the antigens content (HIV-1 antigen P1 and rgp41) weremonitored for degradations over 3 months using HPLC assay). For theliquid vaccine MYM V202 (starting material), it was found that that theP1 content was reduced by 4% and 7% when stored at 2-8° C. (cold storagecondition) for 1 month and 3 months respectively. For rgp41, the contentwas reduced by 16% and 25% at 1 and 3 months respectively at coldstorage condition. When the liquid virosome was stored at 25° C. and 40°C., P1 and rgp41 were no longer detected after 1 month and 3 months.

The results of the antigen contents in the lyophilised tablet on storageare presented in Table 15 and Table 16 for antigen P1 and antigen rgp41respectively. In the lyophilised tablet form, the P1 antigen remainedvery stable without observed content decline after 3 months at 2-8° C.(cold chain condition) and also remained in unaffected during 1 and 3months storage outside the cold chain condition. For the rgp41 antigen,a decline of about 5%, 10% and 20% was observed after 1 months at 2 −8°C., 25° C. and 40° C. respectively. At 3 months storage, the decline wasabout 13%, 15% and 19% respectively. Taken into consideration of theHPLC method accuracy, an observed decline concentration difference ofless the 15% are not significant. Furthermore, the observed gradualantigen loss or decline is related to chemical modifications and not dueto advanced degradation with structural cleavage, amino acid loses oraggregation. Meanwhile, chemical modification(s) in a given epitope maypotentially alter its recognition, decreasing or increasing antibodiesbinding toward that region, while other regions would remain equallywell recognized.

At T0, P1 and rgp41 antigens harbour similar SDS-PAGE migration profileand were still recognized by specific monoclonal antibodies, once undersublingual tablets, and serum antibodies toward P1 and rgp41 were stillreacting toward various P1 and gp41 peptides harbouring key epitopes.These analyses suggest that in overall, P1 and rgp41 have preserved mostof their antigenicity and immunogenicity during the manufacturingprocess (data not shown).

TABLE 15 P1 content (lyophilized tablet) Time point μg/ml μg/unit %reduction/increase Storage at 2-8° C. (average 5° C.) (cold chaincondition) Intitial (T0) 24.7 12.35 Not applicable 1 month (T1) 28.614.3 +15.8 (no lost) 3 months (T3) 27.6 13.8 +11.7 (no lost) Storage at25° C./60% RH 1 month (T1) 27.9 13.95 +13.0 (no lost) 3 months (T3) 27.513.75 +11.13 (no lost) Storage at 40° C./75% RH 1 month (T1) 25.8 12.9+4.5 (no lost) 3 months (T3) 24.0 12.0 −2.8 (lost)

TABLE 16 rgp41 content (lyophilized tablet) Time point μg/ml μg/unit %reduction Storage at 2-8° C. (average 5° C.) (cold chain condition)Initial (T0) 13.5 6.75 Not applicable 1 month (T1) 12.8 6.4 −5.2 3months (T3) 11.8 5.9 −12.6 Storage at 25° C./60% RH 1 month (T1) 12.16.05 −10.4 3 months (T3) 11.5 5.75 −14.8 Storage at 40° C./75% RH 1month (T1) 10.8 5.4 −20.0 3 months (T3) 10.9 5.45 −19.3

Example 4: Stability of Lyophilized Tablets Stored Under Sub-ZeroTemperature Storage Conditions

The data in Example 4 shows the stability of the physicalcharacteristics of the virosome vaccine tablets and the virosomeparticles when stored under sub-zero conditions. A liquid virosomeformulation mix containing 25% w/w loading of liquid virosomesconcentrate MYM-201 lot 160125-1 supplied by Mymetics (containing HA70-80 μg/ml, P1 40-50 μg/ml, rgp41 70-80 μg/ml (A/Brisbane/59/2007(H1N1) in HN buffer pH 7.4 (50 mM HEPES, 142.5 mM NaCl), 6% w/w fishgelatin, 8% w/w mannitol, and 2% w/w (net) trehalose was dosed with a500 mg dosing fill weight and freeze dried. The formulations were dosedwith a 500 mg dosing fill weight aliquot at 15° C. into blister pocketsof aluminum trays. The trays containing the dosed aqueous vaccine mixwere frozen by passing the aluminium trays through a freezing chamberset at −70° C. for a duration of 3 minutes 15 seconds. The aluminiumtrays containing the frozen products were collected and placed in afreezer at a temperature of <−15° C. and annealed for 6 hours frozenhold before lyophilisation. A 2-step FD cycle using −15° C. for 24 hoursfollowed by −10° C. for 18 hours was then used. The blisters offreeze-dried tablets were sealed in sachets and were placed on storagein a freezer at −15° C. for 1 week. A corresponding set of blisters oftablets were sealed in sachets and were placed at ambient conditions forthe same durations. These tablets were assessed for appearance,dispersion characteristics (wetting time and dissociation time) andvirosome particle size distribution as described according to the testmethods above.

All units were found to have good appearance after storage at bothconditions. The data showed that sub-zero storage had little effect onthe tablet dispersion characteristics (wetting and dissociation times)(Table 17) and virosome particle size distribution (Table 18).

TABLE 17 Condition Wetting Time (sec) Dissociation Time (sec) AmbientStorage <31 <37 Sub-Zero Storage <32 <35

TABLE 18 NTA: % NTA: Main Peak virosome Particle Size particle Condition(nm) (<200 nm) 1 week ambient 130 59.9 Post-Digestion 1 week Sub- 13260.6 Zero, Post-Digestion

Example 5: Flow Cytometry Assessment of Virosome Particles fromReconstituted Freeze-Dried Tablets

Estimations of virosome proportions preserved in freeze-dried tabletsamples from process development is summarised below. The valuesprovided in Table 19 were derived from AMNIS flow cytometry data usingmeasurements of events in the focus area corresponding to the virosomegates.

TABLE 19 Percentage of Percentage of virosome recovered clusters(doublets, Vaccine from end of triplets, higher formulation productionforms) Liquid vaccine 100% <5% Freeze-dried tablet 24-40% 10-25%(reconstitutes

The data shows that 24-40% of the starting virosome is preserved in thefreeze-dried tablets of which between 10-25% of these virosomes are inclusters, mostly as doublets and triplets.

Example 6: Immunogenicity Evaluation of P1 and Rgp41 Antigens fromLiquid Virosome Formulation and Reconstituted Freeze Dried SublingualTablets Containing Virosome

Liquid virosome concentrates (liquid vaccine MYM-V202) and freeze-driedsublingual tablets containing virosome for Example 3 and placed onstorage at 4° and 40° C. over a period of three months forimmunogenicity assessment. After storage for 1 month, samples of theliquid vaccine and sublingual tablets stored at 40° C. were removed forthe storage cabinet for immunogenicity assessment. After storage for 3months, samples of the liquid vaccine and sublingual tablets stored at4° C. and 40° C. were removed for immunogenicity assessment.

For the immunogenicity assessment, Wistar rats (n=10 per group), 50% ofeach gender were used. The rats were immunized at day 0, day 28 and day56. The liquid vaccine contained 3.9 μg of P1, 2.2 μg of rgp41 and 1.3μg of 3M-052 TLR7/8 (adjuvant) in 0.1 mL and this was used forsubcutaneous injection. For the sublingual tablets, an adequate quantityof sublingual tablet was dissolved in sterile water to achieve about 3μg of P1, 1.7 μg of rgp41 and 1 μg of 3M-052 TLR7/8 (adjuvant) in 0.1 mLto be administered by subcutaneous injection. Pre-immune serums werecollected at day 0 and immune serums at day 65 for determining the endpoint antibody titers for each animal serums and on the serum pool.

FIG. 5 shows the immunogenicity of P1 and rgp41 from the liquid vaccineand sublingual tablets stored at different temperatures. The liquidadjuvanted vaccine formulation MYM-V202 containing both P1 and rgp41antigens were temperature sensitive and served as reference material forcomparison with the immunogenicity of the sublingual tablet vaccine formwith improve thermostability. (Black line) shows vaccines stored for 3months at 4° C.; (Black dash line) shows vaccine stored for 1 month at40° C.; (Grey line) shows vaccines stored for 3 months at 40° C. Thedata shows that the immunogenicity of the antigens for the freeze-driedtablets are retained. In each panel, the endpoint antibody titers arealso indicated. End point titer corresponds to the last serum dilutiongenerating an OD value >2-fold above the pre-immune background.

Additional Definitions

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”. In addition, reference to phrases “less than”, “greater than”,“at most”, “at least”, “less than or equal to”, “greater than or equalto”, or other similar phrases followed by a string of values orparameters is meant to apply the phrase to each value or parameter inthe string of values or parameters. For example, a statement that asolution has a concentration of at least about 10 mM, about 15 mM, orabout 20 mM is meant to mean that the solution has a concentration of atleast about 10 mM, at least about 15 mM, or at least about 20 mM.

As used herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It is also to be understood that the term “and/or” as usedherein refers to and encompasses any and all possible combinations ofone or more of the associated listed items. It is further to beunderstood that the terms “includes, “including,” “comprises,” and/or“comprising,” when used herein, specify the presence of stated features,integers, steps, operations, elements, components, and/or units but donot preclude the presence or addition of one or more other features,integers, steps, operations, elements, components, units, and/or groupsthereof.

This application discloses several numerical ranges in the text andfigures. The numerical ranges disclosed inherently support any range orvalue within the disclosed numerical ranges, including the endpoints,even though a precise range limitation is not stated verbatim in thespecification because this disclosure can be practiced throughout thedisclosed numerical ranges.

The above description is presented to enable a person skilled in the artto make and use the disclosure, and is provided in the context of aparticular application and its requirements. Various modifications tothe preferred embodiments will be readily apparent to those skilled inthe art, and the generic principles defined herein may be applied toother embodiments and applications without departing from the spirit andscope of the disclosure. Thus, this disclosure is not intended to belimited to the embodiments shown, but is to be accorded the widest scopeconsistent with the principles and features disclosed herein.

The invention claimed is:
 1. An oral solid vaccine dosage formcomprising: lipid-based vesicles comprising an immunogenic amount of atleast one target molecule; 5-20 wt. % of at least onecryo-lyoprotectant; 25-40 wt. % of a matrix former; and 40-55 wt. % of astructure former.
 2. The dosage form of claim 1, wherein the lipid-basedvesicles are virosomes or proteoliposomes.
 3. The dosage form of claim1, wherein the dosage form comprises 10-15 wt. % of at least onecryo-lyoprotectant.
 4. The dosage form of claim 1, wherein the at leastone cryo-lyoprotectant comprises trehalose.
 5. The dosage form of claim1, wherein the dosage form comprises 33-37 wt. % of the matrix former.6. The dosage form of claim 1, wherein the matrix former comprisesgelatin.
 7. The dosage form of claim 6, wherein the gelatin comprisesfish gelatin.
 8. The dosage form of claim 7, wherein the fish gelatin ishigh molecular weight fish gelatin.
 9. The dosage form of claim 1,wherein the dosage form comprises 45-50 wt. % of the structure former.10. The dosage form of claim 1, wherein the structure former comprisesmannitol.
 11. The dosage form of claim 1, wherein the virosomes arederived from the influenza virus membrane or other enveloped viruses.12. The dosage form of claim 1, wherein the at least one target moleculeis present on the virosome.
 13. The dosage form of claim 1, wherein theat least one target molecule comprises an HIV-1 envelope derivedantigen.
 14. The dosage form of claim 13, wherein the HIV-1 envelopederived antigen comprises HIV-1 PI peptide and/or HIV-1 recombinantgp41.
 15. The dosage form of claim 1, wherein the virosomes compriseadjuvant.
 16. The dosage form of claim 1, wherein the dosage form isconfigured to disintegrate in an oral cavity to facilitate oral cavityuptake of the at least one target molecule.
 17. The dosage form of claim16, wherein the dosage form is configured to disintegrate within 180seconds after being placed in the oral cavity.
 18. The dosage form ofclaim 16, wherein the dosage form is configured to disintegrate within90 seconds after being placed in the oral cavity.
 19. The dosage form ofclaim 16, wherein the dosage form is configured to disintegrate within60 seconds after being placed in the oral cavity.
 20. The dosage form ofclaim 16, wherein the dosage form is configured to disintegrate within30 seconds after being placed in the oral cavity.
 21. The dosage form ofclaim 16, wherein the dosage form is configured to induce an immuneresponse when administered to a patient by placement in the oral cavity.22. The dosage form of claim 21, wherein placement in the oral cavity isplacement on or under the tongue or in the buccal or pharyngeal region.23. A method of inducing an immune response in a patient, the methodcomprising placing the dosage form of claim 1 in an oral cavity of aperson in need of the immune response.
 24. The method of claim 23,wherein placement in the oral cavity is placement on or under the tongueor in the buccal or pharyngeal region.
 25. A method of forming an oralsolid vaccine dosage form, the method comprises: dosing a liquidvirosome formulation into a preformed mold, wherein the virosomeformulation comprises: lipid-based vesicles comprising an immunogenicamount of at least one target molecule; 1-5 wt. % of acryo-lyoprotectant; 4-8 wt. % of a matrix former; and 5-10 wt. % of astructure former; freezing the dosed virosome formulation at atemperature of −60° C. to −90° C.; annealing the frozen virosomeformulation by holding it at a temperature of less than −15° C. for 3-9hours; and freeze-drying the annealed virosome formulation to form thedosage form.
 26. The method of claim 25, wherein the dosed virosomeformulation is frozen at a temperature of −60° C. to −90° C. for aduration of about 1-5 minutes.
 27. The method of claim 25, whereinfreeze-drying the annealed virosome formulation comprises a first stepof holding the annealed virosome formulation at a temperature of −10° C.to −20° C. for 20-28 hours and a second step of holding the annealedvirosome formulation at a temperature of −5° C. to about −15° C. for14-22 hours.
 28. The method of claim 25, wherein the freeze-dryingoccurs at a pressure of less than 600 mbar.
 29. The method of claim 25,wherein the virosome formulation has a pH of about 6.5-8.
 30. The methodof claim 25, wherein the cryo-lyoprotectant comprises trehalose.
 31. Themethod of claim 25, wherein the matrix former comprises gelatin.
 32. Themethod of claim 31, wherein the gelatin comprises fish gelatin.
 33. Themethod of claim 32, wherein the fish gelatin is high molecular weightfish gelatin.
 34. The method of claim 25, wherein the structure formercomprises mannitol.
 35. The method of claim 25, wherein the lipid-basedvesicles are derived from the influenza virus or respiratory syncytialvirus.
 36. The method of claim 25, wherein the at least one targetmolecule comprises an HIV-1 envelope derived antigen.
 37. The method ofclaim 36, wherein the HIV-1 envelope derived antigen comprises HIV-1 PIpeptide and/or HIV-1 recombinant gp41.
 38. The method of claim 25,wherein the lipid-based vesicles comprise adjuvant.
 39. A method offorming an oral solid vaccine dosage form, the method comprises: dosinga liquid virosome formulation into a preformed mold, wherein thevirosome formulation comprises: 20-50 wt. % of a virosome concentrate,wherein the virosome concentrate comprises: virosomes comprising animmunogenic amount of at least one target molecule; 2-10 wt. % of acryo-lyoprotectant; and 60-200 mM of a buffer system; 4-8 wt. % of amatrix former; and 5-10 wt. % of a structure former; freezing the dosedvirosome formulation at a temperature of −60° C. to −80° C.; annealingthe frozen virosome formulation by holding it at a temperature of lessthan −15° C. for 3-9 hours; and freeze-drying the annealed virosomeformulation to form the dosage form.
 40. The method of claim 39, whereinthe buffer system comprises HEPES-Sodium Chloride.